AU2008255266B2 - Compositions for use in identification of bacteria - Google Patents

Abstract

COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA Abstract The present invention provides oligonucleotide primers and compositions and kits containing the same for rapid identification of bacteria by amplification of a segment of s bacterial nucleic acid followed by molecular mass analysis.

Description

S&F Ref: 780322D1 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address Ibis Biosciences, Inc., of 1896 Rutherford Road, of Applicant : Carlsbad, California, 92008, United States of America Actual Inventor(s): David J. Ecker Mark W. Eshoo Thomas A. Hall Christian Massire Rangarajan Sampath Address for Service: Spruson & Ferguson St Martins Tower Level 35 31 Market Street Sydney NSW 2000 (CCN 3710000177) Invention Title: Compositions for use in identification of bacteria The following statement is a full description of this invention, including the best method of performing it known to me/us: 5845c(1890676_1) -1 COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS [00011 The present application claims the benefit of priority to: U.S. Provisional Application . Serial No. 60/545,425 filed February 18,2004, U.S. Provisional Application Serial No. 60/559,754, filed April 5, 2004, U.S. Provisional Application Serial No. 60/632,862, filed December 3, 2004, U.S. Provisional Application Serial No. 60/639,068, filed December 22, 2004, and U.S. Provisional Application Serial No. 60/648,188, filed January 28, 2005, each of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT [0002] This invention was made with United States Government support under DARPA/SPO contract BAAOO-09. The United States Government may have certain rights in the invention. FIELD OF THE INVENTION [00031 The present invention relates generally to the field of genetic identification of bacteria and provides nucleic acid compositions and kits useful for this purpose when combined with molecular mass analysis. BACKGROUND OF THE INVENTION [0004] A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans. s R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals. [00051 Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific 0 primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of 2 bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture. 5 100061 A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need. Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities 1o of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive. 100071 Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. is DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism. 20 [00081 There is a need for a method for identification of bioagents which is both specific and rapid, and in which no culture or nucleic acid sequencing is required. Disclosed in U.S. Patent Application Serial Nos: 09/798,007 (U.S. Pat. Pub. 20030027135), 09/891,793 (U.S. Pat. 7,217,510), 10/405,756 (U.S. Pat. Pub. 20030228571), 10/418,514 (U.S. Pat. Pub. 20040209260), 10/660,997 (U.S. Pat. 7,226,739), 10/660,122 (U.S. Pat. 25 Pub. 20040219517), 10/660,996 (U.S. Pat. 7,255,992), 10/728,486, 10/754,415 and 10/829,826 (U.S. Pat. Pub. 20050266397), each of which is commonly owned and incorporated herein by reference in its entirety, are methods tor identification of bioagents (any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus) in an unbiased manner by molecular mass and base composition 30 analysis of "bioagent identifying amplicons" which are obtained by amplification of segments of essential and conserved genes which are involved in, for example, translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins include, but are not limited to, ribosomal RNAs, 3 ribosomal proteins, DNA and RNA polymerases, elongation factors, tRNA synthetases, protein chain initiation factors, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, DNA gyrases and DNA topoisomerases, metabolic enzymes, and the like. 5 100091 To obtain bioagent identifying amplicons, primers are selected to hybridize to conserved sequence regions which bracket variable sequence regions to yield a segment of nucleic acid which can be amplified and which is amenable to methods of molecular mass analysis. The variable sequence regions provide the variability of molecular mass 10 which is used for bioagent identification. Upon amplification by PCR or other amplification methods with the specifically chosen primers, an amplification product that represents a bioagent identifying amplicon is obtained. The molecular mass of the amplification product, obtained by mass spectrometry for example, provides the means to uniquely identify the bioagent without a requirement for prior knowledge of the possible is identity of the bioagent. The molecular mass of the amplification product or the corresponding base composition (which can be calculated from the molecular mass of the amplification product) is compared with a database of molecular masses or base compositions and a match indicates the identity of the bioagent. Furthermore, the method can be applied to rapid parallel analyses (for example, in a multi-well plate format) the 20 results of which can be employed in a triangulation identification strategy which is amenable to rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent identification. 100101 The result of determination of a previously unknown base composition of a 25 previously unknown bioagent (for example, a newly evolved and heretofore unobserved bacterium or virus) has downstream utility by providing new bioagent indexing information with which to populate base composition databases. The process of subsequent bioagent identification analyses is thus greatly improved as more base composition data for bioagent identifying amplicons becomes available. 30 [00111 The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose 3a molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level. SUMMARY OF THE INVENTION 5 [001 Ia] According to a first aspect of the invention there is provided a composition comprising an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 350. io [001 1b] According to a second aspect of the invention there is provided the composition of the first aspect, further comprising a second oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 712. is [001lc] According to a third aspect of the invention there is provided a method for identification of an unknown bacterium comprising: amplifying nucleic acid from said bacterium using the composition of the second aspect to obtain an amplification product; determining the molecular mass of said amplification product; 20 optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with a plurality of molecular masses or base compositions of known bacterial bioagent identifying amplicons, wherein a match between said molecular mass or base 25 composition of said amplification product and the molecular mass or base composition of a member of said plurality of molecular masses or base compositions identifies said unknown bacterium. [0011 d] According to a fourth aspect of the invention there is provided a method of 30 determining the presence or absence of a bacterium of a particular clade, genus, species, or sub-species in a sample comprising: amplifying nucleic acid from said sample using the composition of the second aspect to obtain an amplification product; determining the molecular mass of said amplification product; 3b optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with the known molecular masses or base compositions of one or more known 5 clade, genus, species, or sub-species bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of one or more known clade, genus, species, or sub species bioagent identifying amplicons indicates the presence of said clade, genus, species, or sub-species in said sample. 10 [0011 e) According to a fifth aspect of the invention there is provided a method for determination of the quantity of an unknown bacterium in a sample comprising: contacting said sample with the composition of the second aspect and a known quantity of a calibration polynucleotide comprising a calibration sequence; 15 concurrently amplifying nucleic acid from said bacterium in said sample with the composition of the second aspect and amplifying nucleic acid from said calibration polynucleotide in said sample with the composition of the second aspect to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon; 20 determining the molecular mass and abundance for said bacterial bioagent identifying amplicon and said calibration amplicon; and distinguishing said bacterial bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the 25 quantity of bacterium in said sample. 10012] The present invention provides primers and compositions comprising pairs of primers, and kits containing the same for use in identification of bacteria. The primers are designed to produce bacterial bioagent identifying amplicons of DNA encoding genes essential to life such as, for example, 16S and 23S rRNA, DNA-directed RNA 30 polymerase subunits (rpoB and rpoC), -4 valyl-tRNA synthetase (valS), elongation factor EF-Tu (TufB), ribosomal protein L2 (rplB), protein chain initiation factor (infB), and spore protein (sspE). The invention further provides drill-down primers, compositions comprising pairs of primers and kits containing the same, which are designed to provide sub-species characterization of bacteria. S [0013] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 97, or a composition comprising the same; an oligonucleotide primer 20 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 451, or a composition comprising the same; a 10 composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 97, and a second oligonucleotide primer 20 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 451. 15 [0014] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 127, or a composition comprising the same; an oligonucleotide primer 14 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 482, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide )o primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 127, and a second oligonucleotide primer 14 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 482. [0015] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in ,5 length comprising 70% to 100% sequence identity with SEQ ID NO: 174, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 530, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of 30 SEQ ID NO: 174, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 530. [0016] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 310, or a composition comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 668, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of S SEQ ID NO: 310, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 668. [0017] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 313, or a composition o comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 670, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 313, and a second oligonucleotide primer 21 to 35 nucleobases in length i comprising between 70% to 100% sequence identity of SEQ ID NO: 670. [0018] The present invention also provides an oligonucleotide primer 17 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 277, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% y to 100% sequence identity with SEQ ID NO: 632, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 17 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 277, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 632. [0019] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 285, or a composition comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 640, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 285, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 640.

-6 [0020] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 301, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 656, or a composition comprising the same; a s composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 301, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 656. [0021] The present invention also provides an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 308, or a composition comprising the same; an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 663, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide s primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 308, and a second oligonucleotide primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 663. [0022] The present invention also provides compositions, such as those described herein, o wherein either or both of the first and second oligonucleotide primers comprise at least one modified nucleobase, a non-templated T residue on the 5'-end, at least one non-template tag, or at least one molecular mass modifying tag, or any combination thereof. [0023] The present invention also provides kits comprising any of the compositions described 2- herein. The kits can comprise at least one calibration polynucleotide, or at least one ion exchange resin linked to magnetic beads, or both. [0024] The present invention also provides methods for identification of an unknown bacterium. Nucleic acid from the bacterium is amplified using any of the compositions described herein to I obtain an amplification product The molecular mass of the amplification product is determined. Optionally, the base composition of the amplification product is determined from the molecular mass. The base composition or molecular mass is compared with a plurality of base compositions or molecular masses of known bacterial bioagent identifying amplicons, wherein a match between the base composition or molecular mass and a member of the plurality of base compositions or molecular masses identifies the unknown bacterium. The molecular mass can be measured by mass spectrometry. In addition, the presence or absence of a particular clade, genus, species, or sub-species of a bioagent can be determined by the methods described herein. 5 [00251 The present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with any of the compositions described herein and a known quantity of a calibration polynucleotide comprising a calibration sequence. Concurrently, nucleic acid from the bacterium in the sample is amplified with any of the compositions described herein and nucleic acid from the calibration polynucleotide in the sample io is amplified with any of the compositions described herein to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular mass and abundance for the bacterial bioagent identifying amplicon and the calibration amplicon is determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass, 5 wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. The method can also comprise determining the base composition of the bacterial bioagent identifying amplicon. BRIEF DESCRIPTION OF THE DRAWINGS [00261 Figure 1 is a represenataive pseudo-four dimensional plot of base compositions of bioagent identifying amplicons of enterobacteria obtained with a primer pair targeting the rpoB gene (primer pair no 14 (SEQ ID NOs: 37:362). The quantity each of the nucleobases A, G and C are represented on the three axes of the plot while the quantity of nucleobase T is represented by the diameter of the spheres. Base composition probability clouds surrounding the spheres are 5 also shown. [0027] Figure 2 is a represenataive diagram illustrating the primer selection process. [0028] Figure 3 lists common pathogenic bacteria and primer pair coverage. The primer pair o) number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon. [0029] Figure 4 is a represenataive 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair -8 number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms. 5 [00301 Figure 5 is a represenataive mass spectrum of amplification products representing bioagent identifying amplicons of Streptococcuspyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses o and base compositions for the sense strand of each amplification product are shown. [00311 Figure 6 is a represenataive mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which 15 targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown. [0032] Figure 7 is a represenataive process diagram for identification and determination of the quantity of a bioagent in a sample. ro [0033] Figure 8 is a represenataive mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 741), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the as amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis. DESCRIPTION OF EMBODIMENTS [0034] The present invention provides oligonucleotide primers which hybridize to conserved ,o regions of nucleic acid of genes encoding, for example, proteins or RNAs necessary for life which include, but are not limited to: 16S and 23S rRNAs, RNA polymerase subunits, t-RNA synthetases, elongation factors, ribosomal proteins, protein chain initiation factors, cell division proteins, chaperonin groEL, chaperonin dnaK, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, metabolic enzymes and DNA topoisomerases. These primers provide the -9 functionality of producing, for example, bacterial bioagent identifying amplicons for general identification of bacteria at the species level, for example, when contacted with bacterial nucleic acid under amplification conditions. 5 [0035] Referring to Figure 2, primers are designed as follows: for each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are designed by selecting appropriate priming regions (230) which allows the selection of candidate primer pairs (240). The primer pairs are subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are o obtained from sequence databases such as, for example, GenBank or other sequence collections (310), and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a particular amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are stored in a base composition 15 database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (240) are validated by in vitro amplification by a method such as, for example, PCR analysis (400) of nucleic acid from a collection of organisms (410). Amplification products that are obtained are optionally analyzed to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420). [00361 Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied >S Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. [0037] The primers can be employed as compositions for use in, for example, methods for identification of bacterial bioagents as follows. In some embodiments, a primer pair composition o is contacted with nucleic acid of an unknown bacterial bioagent. The nucleic acid is amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of one strand or each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as, for example, mass spectrometry wherein the two strands of the -10 double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF MS). A list of possible base compositions can be generated for the molecular mass value 6 obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known bacterial bioagents. A match between the molecular mass or base composition of the 10 amplification product from the unknown bacterial bioagent and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known bacterial bioagent indicates the identity of the unknown bioagent. [0038] In some embodiments, the primer pair used is one of the primer pairs of Table 1. In some IS embodiments, the method is repeated using a different primer pair to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment. [00391 In some embodiments, a bioagent identifying amplicon may be produced using only a )o single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation. [0040] In some embodiments, the oligonucleotide primers are "broad range survey primers" which hybridize to conserved regions of nucleic acid encoding RNA, such as ribosomal RNA (rRNA), of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of known bacteria and produce bacterial bioagent identifying amplicons. As used herein, the term "broad ?o range survey primers" refers to primers that bind to nucleic acid encoding rRNAs of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% known species of bacteria. In some embodiments, the rRNAs to which the primers hybridize are 16S and 23S rRNAs. In some embodiments, the broad range survey primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer -11 pair numbers 3, 10, 11, 14, 16, and 17 which consecutively correspond to SEQ ID NOs: 6:369, 26:388, 29:391, 37:362, 48:404, and 58:414. [0041] In some cases, the molecular mass or base composition of a bacterial bioagent identifying 5 amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional "division-wide" primer pair (vide infra). The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as "triangulation identification" (vide infra). [00421 In other embodiments, the oligonucleotide primers are "division-wide" primers which hybridize to nucleic acid encoding genes of broad divisions of bacteria such as, for example, members of the Bacillus/Clostridia group or members of the a-, p-, y-, and e-proteobacteria. In 5 some embodiments, a division of bacteria comprises any grouping of bacterial genera with more than one genus represented. For example, the P-proteobacteria group comprises members of the following genera: Eikenella, Neisseria, Achromobacter, Bordetella, Burkholderia, and Raltsonia. Species members of these genera can be identified using bacterial bioagent identifying amplicons generated with primer pair 293 (SEQ ID NOs: 344:700) which produces a bacterial bioagent identifying amplicon from the tufB gene of fp-proteobacteria. Examples of genes to which division-wide primers may hybridize to include, but are not limited to: RNA polymerase subunits such as rpoB and rpoC, tRNA synthetases such as valyl-tRNA synthetase (valS) and aspartyl-tRNA synthetase (aspS), elongation factors such as elongation factor EF-Tu (tufB), ribosomal proteins such as ribosomal protein L2 (rplB), protein chain initiation factors such as 5 protein chain initiation factor infB, chaperonins such as groL and dnaK, and cell division proteins such as peptidase fisH (hflB). In some embodiments, the division-wide primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair numbers 34, 52, 66, 67, 71, 72, 289, 290 and 293 which consecutively correspond to SEQ ID NOs: 160:515, 261:624, 231:591, 235:587, to 349:711, 240:596, 246:602, 256:620, 344:700. [0043] In other embodiments, the oligonucleotide primers are designed to enable the identification of bacteria at the clade group level, which is a monophyletic taxon referring to a group of organisms which includes the most recent common ancestor of all of its members and -12 all of the descendants of that most recent common ancestor. The Bacillus cereus clade is an example of a bacterial clade group. In some embodiments, the clade group primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair number 58 which corresponds to SEQ ID NOs: 3i 322:686. [00441 In other embodiments, the oligonucleotide primers are "drill-down" primers which enable the identification of species or "sub-species characteristics." Sub-species characteristics are herein defined as genetic characteristics that provide the means to distinguish two members of the same bacterial species. For example, Escherichia coli 0157:H7 and Escherichia coli KI 2 are two well known members of the species Escherichia coli. Escherichia coli 0157:H7, however, is highly toxic due to the its Shiga toxin gene which is an example of a sub-species characteristic. Examples of sub-species characteristics may also include, but are not limited to: variations in genes such as single nucleotide polymorphisms (SNPs), variable number tandem repeats 5 (VNTRs). Examples of genes indicating sub-species characteristics include, but are not limited to, housekeeping genes, toxin genes, pathogenicity markers, antibiotic resistance genes and virulence factors. Drill-down primers provide the functionality of producing bacterial bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with bacterial nucleic acid under amplification conditions. Identification of such sub-species characteristics is o often critical for determining proper clinical treatment of bacterial infections. Examples of pairs of drill-down primers include, but are not limited to, a trio of primer pairs for identification of strains of Bacillus anthracis. Primer pair 24 (SEQ ID NOs: 97:45 1) targets the capC gene of virulence plasmid pX02, primer pair 30 (SEQ ID NOs: 127:482) targets the cyA gene of virulence plasmid pX02, and primer pair 37 (SEQ ID NOs: 174:530) targets the lef gene of 2,5 virulence plasmid pX02. Additional examples of drill-down primers include, but are not limited to, six primer pairs that are used for determining the strain type of group A Streptococcus. Primer pair 80 (SEQ ID NOs: 310:668) targets the gki gene, primer pair 81 (SEQ ID NOs: 313:670) targets the gtr gene, primer pair 86 (SEQ ID NOs: 227:632) targets the murl gene, primer pair 90 (SEQ ID NOs: 285:640) targets the mutS gene, primer pair 96 (SEQ ID NOs: v 301:656) targets the xpt gene, and primer pair 98 (SEQ ID NOs: 308:663) targets the yqiL gene. [0045] In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, DNA of bacterial plasmids, or DNA of DNA viruses.

- 13 [0046] In some embodiments, the primers used for amplification hybridize directly to ribosomal RNA or messenger RNA (mRNA) and act as reverse transcription primers for obtaining DNA from direct amplification of bacterial RNA or rRNA. Methods of amplifying RNA using reverse transcriptase are well known to those with ordinary skill in the art and can be routinely established without undue experimentation. [00471 One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a o primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 1. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, 5, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20 0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20 = 0.75 or 75% sequence identity with the 20 nucleobase primer. [00481 Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics as Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, homology, sequence identity, or complementarity of primers with respect to the conserved priming regions of bacterial nucleic acid, is at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%. 0 [0049] In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein. Thus, for example, a primer may have between 70% and -14 100%, between 75% and 100%, between 80% and 100%, and between 95% and 100% sequence identity with SEQ ID) NO: 26. Likewise, a primer may have similar sequence identity with any other primer whose nucleotide sequence is disclosed herein. [00501 One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon. lo [0051] In some embodiments of the present invention, the oligonucleotide primers are between 13 and 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin. 1S [0052] In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5' end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated A residues as a result of the 10 non-specific enzyme activity of Taq polymerase (Magnuson et al. Biotechniques, 1996, 21, 700 709), an occurrence which may lead to ambiguous results arising from molecular mass analysis. [0053] In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3 P position) in the conserved as regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal nucleobase." For example, under this "wobble" pairing, inosine (1) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053 1056) or the purine analog 1-(2-deoxy-p.-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

15 100541 In some embodiments, to compensate for the somewhat weaker binding by the "wobble" base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not s limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil which binds to adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Patent Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S Serial No. 10/294,203 (U.S. Pat. Pub. 10 20030170680) which is also commonly owned and incorporated herein by reference in entirety. Phenoxazines are described in U.S. Patent Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Patent Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety. 15 10055] In some embodiments, non-template primer tags are used to increase the melting temperature (T m) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A 20 can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to a A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles. 25 100561 In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified intenucleoside linkage such as a phosphorothioate linkage, for 30 example. [0057] In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base 35 composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon (vide infra) from its molecular mass. 3382844_L.DOC:LNB - 16 [00581 In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2' deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2' deoxycytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2' deoxyuridine-5'-triphosphate, 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5' triphosphate, 5-fluoro-2'-deoxyuridine-5'-triphosphate, 06-methyl-2'-deoxyguanosine-5' triphosphate, N2-methyl-2'-deoxyguanosine-5'-triphosphate, 8-oxo-2'-deoxyguanosine-5' triphosphate or thiothymidine-5'-triphosphate. In some embodiments, the mass-modified nucleobase comprises "N or "C or both 15N and "C. 0 [0059] In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bioagent. Thus,,the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass 5 determination are herein described as "bioagent identifying amplicons." The term "amplicon" as used herein, refers to a segment of a polynucleotide which is amplified in an amplification reaction. In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 200 nucleobases (i.e. from about 45 to about 200 linked nucleosides), from about 60 to about 150 nucleobases, from about 75 to about 125 nucleobases. 0 One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119,120,121,122,123,124,125,126,127,128,129,130,131, 132,133,134,135,136, -.5 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 nucleobases in length, or any range therewithin. It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize 30 (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is prudent to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.

17 100601 In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to s obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with restriction enzymes or cleavage primers, for example. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art. 10 [00611 In some embodiments, amplification products corresponding to bacterial bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) which is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency 15 single primer PCR, and multiple strand displacement amplification (MDA) which are also well known to those with ordinary skill. 100621 In the context of this invention, a "bioagent" is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of 20 bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or 25 spores) and may be encapsulated or bioengineered. In the context of this invention, a "pathogen" is a bioagent which causes a disease or disorder. [00631 In the context of this invention, the term "unknown bioagent" may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species 30 Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of corona viruses disclosed in commonly owned U.S. Patent Serial No. 10/829,826 (U.S. Pat. Pub. 20050266397), (incorporated herein by reference in its 35 entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of "unknown" bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what 18 bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. Patent Serial No. 10/829,826 (U.S. Pat. Pub. 20050266397) was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning (i) of "unknown" bioagent would apply since the SARS 5 coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample. 100641 The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as "triangulation identification". Triangulation io identification is pursued by analyzing a plurality of bioagent identifying amplicons selected within multiple core genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Apple. Microbiol., 1999, 87, 270-278) in the absence of the is expected signatures from the B. anthracis genome would suggest a genetic engineering event. [0065] In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using 20 the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of 25 nucleic acids. [0066] In some embodiments, the molecular mass of a particular bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to 30 separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (m/z). Thus, mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about 35 the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular -19 weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons. 10067] In some embodiments, intact molecular ions are generated from amplification products 5 using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords o an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation. 5 [0068] The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic ,sector, Q-TOF, and triple quadrupole. .o [00691 In some embodiments, conversion of molecular mass data to a base composition is useful for certain analyses. As used herein, a "base composition" is the exact number of each nucleobase (A, T, C and G). For example, amplification of nucleic acid of Neisseria meningitidis with a primer pair that produces an amplification product from nucleic acid of 23S rRNA that has a molecular mass (sense strand) of 28480.75124, from which a base composition of A25 G27 . C22 T18 is assigned from a list of possible base compositions calculated from the molecular mass using standard known molecular masses of each of the four nucleobases. [0070] In some embodiments, assignment of base compositions to experimentally determined molecular masses is accomplished using "base composition probability clouds." Base 30 compositions, like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds" around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" (Figure 1) can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice -20 of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds. [0071] In some embodiments, base composition probability clouds provide the means for screening potential primer, pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for o predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement. aS [00721 The present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent. Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream 20 utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases. [0073] In one embodiment, a sample comprising an unknown bioagent is contacted with a pair as of primers which provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent 30 identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2 to 8 nucleobase deletion or -21 insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular 5 mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample. [0074] In some embodiments, the identity and quantity of a particular bioagent is determined using the process illustrated in Figure 7. For instance, to a sample containing nucleic acid of an unknown bioagent are added primers (500) and a known quantity of a calibration polynucleotide (505). The total nucleic acid in the sample is subjected to an amplification reaction (510) to is obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying o amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample. [0075] In some embodiments, construction of a standard curve where the amount of calibration as polynucleotide spiked into the sample is varied, provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation. 3W [00761 In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the -22 calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation. [00771 In some embodiments, the calibrant polynucleotide is used as an internal positive control s to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such 1o failures have occurred is in itself, a useful event. [0078] In some embodiments, the calibration sequence is inserted into a vector which then itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a is calibration polynucleotide is herein termed a "combination calibration polynucleotide." The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an Lo appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation. [00791 The present invention also provides kits for carrying out, for example, the methods 2S described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer 3 pairs recited in Table 1. [00801 In some embodiments, the kit may comprise one or more broad range survey primer(s), division wide primer(s), clade group primer(s) or drill-down primer(s), or any combination thereof. A kit may be designed so as to comprise particular primer pairs for identification of a - 23 particular bioagent. For example, a broad range survey primer kit may be used initially to identify an unknown bioagent as a member of the Bacillus/Clostridia group. Another example of a division-wide kit may be used to distinguish Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis from each other. A clade group primer kit may be used, for example, to identify an 5 unknown bacterium as a member of the Bacillus cereus clade group. A drill-down kit may be used, for example, to identify genetically engineered Bacillus anthracis. In some embodiments, any of these kits may be combined to comprise a combination of broad range survey primers and division-wide primers, clade group primers or drill-down primers, or any combination thereof, for identification of an unknown bacterial bioagent. 10 [0081] In some embodiments, the kit may contain standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. Patent Application Serial No: 60/545,425 which is incorporated herein by reference in its entirety. 1s [0082] In some embodiments, the kit may also comprise a sufficient quantity of reverse transcriptase (if an RNA virus is to be identified for example), a DNA polymerase, suitable nucleoside triphosphates (including any of those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may 20 further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion a5 exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit. [0083] In order that the invention disclosed herein may be more efficiently understood, examples 30 are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning - A Laboratory Manual, -24 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted. EXAMPLES [00841 Example 1: Selection of Primers That Define Bioagent Identifying Amplicons [0085] For design of primers that define bacterial bioagent identifying amplicons, relevant sequences from, for example, GenBank are obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 200 nucleotides in length and distinguish species from each other by their molecular masses or base compositions. A typical process shown in Figure 2 is employed. [0086] A database of expected base compositions for each primer region is generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nuc. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs. [0087] Table 1 represents a collection of primers (sorted by forward primer name) designed to identify bacteria using the methods herein described. The forward or reverse primer name indicates the gene region of bacterial genome to which the primer hybridizes relative to a reference sequence eg: the forward primer name 1 6SEC_1077_1106 indicates that the primer hybridizes to residues 1077-1106 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence represented by a sequence extraction of coordinates 4033120..4034661 from GenBank gi number 16127994 (as indicated in Table 2). As an additional example: the forward primer name BONTAX52066_450__473 indicates that the primer hybridizes to residues 450 437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 1 are defined in o Table 2). In Table 1, Ua = 5-propynyluracil; Ca = 5-propynylcytosine; * = phosphorothioate linkage. The primer pair number is an in-house database index number. Table 1: Primer Pairs for Identification of Bacterial Bioagents Primer For. For. Rev. pair primer SEQ ID Rev. primer SEQ ID number name Forward sequence NO: Rname everse sequence NO: 1 16S EC 107 GTGAGATGTTGGGTTAA 1 16S EC 1175 GACGTCATCCCCACCTTCC 368 -25 7 1106 F GTCCCGTAACGAG 1195 R TC 16S EC 1177 16S EC 108 ATGTTGGGTTAAGTCCC 1196_OG 1 TGACGTCATGGCCACCTTC 266 2 1100 F GC 2 1G R C 372 16S EC 108 ATGTTGGGTTAAGTCCC 16SEC_1177 TGACGTCATGCCCACCTTC 265 2 1100 F GC 2 1196 1OG R C 373 16S EC 108 ATGTTGGGTTAAGTCCC 16S_EC_1177 TGACGTCATCCCCACCTTC 230 2 1I00~F GC 2 1196 R C 374 16S EC 108 ATGTTGGGTTAAGTCCC 16SEC_1525 263 2 1100 F GC 2 1541 R AAGGAGGTGATCCAGCC 382 16S EC 108 ATGTTGGGTTAAGTCCC 16S EC 1175 TTGACGTCATCCCCACCTT 2 2 1106 F GCAACGAG 3 1197 R CCTC 371 16SEC_109 TTAAGTCCCGCAACGAG 16SEC_1175 TGACGTCATCCCCACCTTC 278 0 1111 2 F CGCAA 4 1196 R CTC 369 16SEC_109 16SEC_1175 0_1111 2_T TTTAAGTCCCGCAACGA 1196_TMOD_ TTGACGTCATCCCCACCTT 361 MOD F GCGCAA 5 R CCTC 370 16SEC_109 TTAAGTCCCGCAACGAT 16_SEC_1175 TGACGTCATCCCCACCTTC 3 0 1111 F CGCAA 6 1196 R CTC 1 369 16_EC_109 TAGTCCCGCAACGAGCG 16SEC 1174 GACGTCATCCCCACCTTCC 256 2 1109 F C 7 1195 R TCC 367 16S_EC_110 16S EC 1174 159 0 1116 F CAACGAGCGCAACCCTT 6 1188 R TCCCCACCTTCCTCC 366 16SEC_119 CAAGTCATCATGGCCCT 16SEC 1525 247 5 1213 F TA 9 1541 R AAGGAGGTGATCCAGCC 382 16SEC_122 GCTACACACGTGCTACA 16SEC_1303 CGAGTTGCAGACTGCGATC 4 2 1241 F ATG 10 1323 R CG 376 16S EC 130 CGGATTGGAGTCTGCAA 16SEC_1389 232 3 1323 F CTCG 11 1407 R GACGGGCGGTGTGTACAAG 378 16S EC 133 AAGTCGGAATCGCTAGT 16S EC_1389 5 2 1353~F AATCG 12 1407 R. GACGGGCGGTGTGTACAAG 378 16SEC_136 TACGGTGAATACGTTCC 16SEC_1485 ACCTTGTTACGACTTCACC 252 7 1387 F CGGG 13 1506 R CCA 379 16SEC_138 GCCTTGTACACACCTCC 16S_EC_1494 CACGGCTACCTTGTTACGA 250 7 1407 F CGTC 14 1513 R C 381 16S EC_138 CTTGTACACACCGCCCG 16S_EC_1525 231 9 1407 F TC 15 1541 R AAGGAGGTGATCCAGCC 382 16SEC_139 TTGTACACACCGCCCGT 16SEC_1486 CCTTGTTACGACTTCACCC 251 0 1411 F CATAC 16 1505 R C , 380 16S_EC_30_ TGAACGCTGGTGGCATG 16S_EC_105_ TACGCATTACTCACCCGTC 6 54 F CTTAACAC 17 126 R CGC 361 16SEC_314 CACTGGAACTGAGACAC 16S EC 556 CTTTACGCCCAGTAATTCC 243 332 F GG 18 5759R ' G 385 16S_EC_38_ GTGGCATGCCTAATACA 16SEC_101_ TTACTCACCCGTCCGCCGC 7 64 F TGCAAGTCG 19 120 R T 357 16SEC_405 TGAGTGATGAAGGCCTT 16SEC 507 CGGCTGCTGGCACGAAGTT 279 432 F AGGGTTGTAAA 20 527 R. AG 384 16SEC_49_ TAACACATGCAAGTCGA 16S EC 104_ 8 68 F ACG 21 120 R TTACTCACCCGTCCGCC 359 16SEC_49_ TAACACATGCAAGTCGA 16SEC_1061 275 68 F ACG 21 1078 R ACGACACGAGCTGACGAC 364 16S_EC_49_ TAACACATGCAAGTCGA 16SEC_880 274 68 F ACG 21 894 R CGTACTCCCCAGGCG 390 16SEC_518 CCAGCAGCCGCGGTAAT 16SEC 774 GTATCTAATCCTGTTTGCT 244 536 F AC 22 795 R CCC 387 16SEC_556 CGGAATTACTGGGCGTA 16SEC 683 226 575 F AAG 23 700 R ' CGCATTTCACCGCTACAC 386 16S EC 556 CGGAATTACTGGGCGTA 16SEC 774 GTATCTAATCCTGTTTGCT 264 573 F AAG 23 795 R CCC 387 16S EC 683 GTGTAGCGGTGAAATGC 16S EC 1303 CGAGTTGCAGACTGCGATC 273 70U F G 24 1323 R CG 377 16S EC_683 GTGTAGCGGTGAAATGC 16SEC 774_ GTATCTAATCCTGTTTGCT 9 700 F G 24 795 R CCC 387 16S EC 683 GTGTAGCGGTGAAATGC 16SEC 880 158 700 F G 24 894 R. CGTACTCCCCAGGCG 390 16S EC 683 GTGTAGCGGTGAAATGC 16SEC_967_ 245 700 F G 24 985 R GGTAAGGTTCTTCGCGTTG 396 16SEC_7_3 GAGAGTTTGATCCTGGC 16SEC 101 TGTTACTCACCCGTCTGCC 294 3 F TCAGAACGAA 25 122 R. ACT 358 16S EC 713 AGAACACCGATGGCGAA 16S EC 789 CGTGGACTACCAGGGTATC 10 732 F GGC 26 8099R TA 388 16S EC 713 732_iTMOD_ TAGAACACCGATGGCGA 16SEC 789 TCGTGGACTACCAGGGTAT 346 F AGGC 27 809 TMOD R CTA 389 228 16S EC 774 GGGAGCAAACAGGATTA 28 16S EC 880 CGTACTCCCCAGGCG 390 -26 795 F GATAC 894 R 16S EC 785 GGATTAGAGACCCTGGT 16S EC 880 11 80ii F AGTCC 29 897-R GGCCGTACTCCCCAGGCG 391 16S EC_785 806_THOD_ TGGATTAGAGACCCTGG 16SEC 880 347 F TAGTCC 30 897 TMOD R TGGCCGTACTCCCCAGGCG 392 16S EC 785 GGATTAGATACCCTGGT 16S EC 880 12 810 F AGTCCACGC 31 897 2 R GGCCGTACTCCCCAGGCG 391 16S EC_789 TAGATACCCTGGTAGTC 16S EC 880 13 810 F CACGC 32 894 R CGTACTCCCCAGGCG 390 16SEC_789 TAGATACCCTGGTAGTC 16S EC 882_ 255 810 F CACGC 32 899 R GCGACCGTACTCCCCAGG 393 16sEC_791 GATACCCTGGTAGTCCA 16SEC 886 254 812 F CACCG 33 904 R. GCCTTGCGACCGTACTCCC 394 16SEC_6_2 AGAGTTTGATCATGGCT 16SEC_1525 248 7 F CAG 34 1541 R AAGGAGGTGATCCAGCC 382 16sEC_8_2 AGAGTTTGATCATGGCT 16SEC 342 242 7 F CAG 34 358 R ACTGCTGCCTCCCGTAG 383 16S_EC_804 ACCACGCCGTAAACGAT 16SEC 909 CCCCCGTCAATTCCTTTGA 253 822 F GA 35 929R GT 395 16S EC_937 AAGCGGTGGAGCATGTG 16SEC_1220 ATTGTAGCACGTGTGTAGC 246 954 F G 36 1240 R CC , 375 16S EC_960 TTCGATGCAACGCGAAG 16SEC_1054 ACGAGCTGACGACAGCCAT 14 981 F AACCT 37 1073 R G 362 16S EC_960 16SEC_1054 _981_TMOD_ TTTCGATGCAACGCGAA _1073_TMOD_ TACGAGCTGACGACAGCCA 348 F GAACCTi 38 R TG 363 16SEC_969 ACGCGAAGAACCTTA 16SEC_1061 119 985 IP F U*C 39 1078 2P R ACGACACGAGU*CaGACGAC 364 165_EC_969 16SEC_1061 15 985 F ACGCGAAGAACCTTACC 39 1078 R9 ACGACACGAGCTGACGAC 364 16SEC_969 16S EC_1389 272 985 F ACGCGAAGAACCTTACC 40 1407 R GACGGGCGGTGTGTACAAG 378 16SEC_971 GCGAAGAACCTTACCAG 16SEC_1043 ACAACCATGCACCACCTGT 344 990 F GTC 41 1062 R C 360 16SEC_972 16SEC_1064 120 985 2P F CGAAGAAUUTTACC 42 1075 2P R ACACGAGU*C'GAC 365 16S EC_972 16SEC_1064 121 985 F CGAAGAACCTTACC 42 1075 R ACACGAGCTGAC 365 23SBRM_11 TGCGCGGAAGATGTAAC 23SBRM_117 TCGCAGGCTTACAGAACGC 1073 10 1129 F GGG 43 6 1201 P. TCTCCTA 397 23S BRM 51 TGCATACAAACAGTCGG 23S_BRM_616 TCGGACTCGCTTTCGCTAC 1074 5 536 F AGCCT 44 635 R G 398 23SBS_- AAACTAGATAACAGTAG 23SBS_5_21 241 68 -44 F ACATCAC 45 R GTGCGCCCTTTCTAACTT 399 23SEC_160 TACCCCAAACCGACACA 23SEC_1686 235 2 1620 F GG 46 1703 R CCTTCTCCCGAAGTTACG 402 23SEC_168 CCGTAACTTCGGGAGAA 23SEC_1828 236 5 1703 F GG 47 1842 R CACCGGGCAGGCGTC 403 23SEC_182 CTGACACCTGCCCGGTG 23SEC_1906 16 6 1843 F C 48 1924 R GACCGTTATAGTTACGGCC 404 23SEC_182 23SEC_1906 6_1843_TMO TCTGACACCTGCCCGGT _1924_TMOD_ TGACCGTTATAGTTACGGC 349 D F GC 49 R C 405 23 SEC_182 23SEC_1929 CCGACAAGGAATTTCGCTA 237 7 1643 F GACGCCTGCCCGGTGC 50 1949 R CC 407 23SEC_183 ACCTGCCCAGTGCTGGA 233_EC_1919 249 1 1849 F AG 51 1936 R TCGCTACCTTAGGACCGT 406 235EC_187 GGGAACTGAAACATCTA 23SEC_242_ 234 207 F AGTA 52 256 R TTCGCTCGCCGCTAC 408 23SEC_23_ 23SEC_115_ 233 37 F GGTGGATGCCTTGGC 53 130 R GGGTTTCCCCATTCGG 401 23SEC_243 AAGGTACTCCGGGGATA 23SEC_2490 AGCCGACATCGAGGTGCCA 238 4 2456 F ACAGGC 54 2511 R AAC 409 23SEC_258 TAGAACGTCGCGAGACA 23SEC_2658 AGTCCATCCCGGTCCTCTC 257 6 2607 F GTTCG 55 2677 R G 411 23SEC_259 GACAGTTCGGTCCCTAT 23SEC_2653 239 9 2616 F C 56 2669 R CCGGTCCTCTCGTACTA 410 23SEC_264 CTGTCCCTAGTACGAGA 23SEC_2751 GTTTCATGCTTAGATGCTT 18 5 2669 2 F GGACCGG 57 2767 R TCAGC 417 23SEC_264 TCTGTCCCTAGTACGAG 23S EC_2744 17 5 2669 F AGGACCGG 58 2761 R TGCTTAGATGCTTTCAGC 414 23SEC_264 CTGTTCTTAGTACGAGA 23 SEC_2745 TTCGTGCTTAGATGCTTTC 118 6 2667 F GGACC 59 2765 P. AG 415 360 235 EC 264 TCTGTTCTTAGTACGAG 60 23S EC 2745 TTTCGTGCTTAGATGCTTT 416 -27 6_2667_TMO AGGACC _2765_TMOD_ CAG D F _ R 23S EC 265 CTAGTACGAGAGGACCG 23S EC 2741 ACTTAGATGCTTTCAGCGG 147 2 2669 F G 61 2760 1 T 413 23S EC 265 23S EC 2737 TTAGATGCTTTCAGCACTT 240 3 2669 F TAGTACGAGAGGACCGG 62 2758 5 ATC 412 23S EC_493 GGGGAGTGAAAGAGATC 23S EC 551 ACAAAAGGCACGCCATCAC 20 518 2 F CTGAAACCG 63 571~2 ~ CC 418 23SEC_493 GGGGAGTGAAAGAGATC 23SEC_551_ ACAAAAGGTACGCCGTCAC 19 518 F CTGAAACCG 63 571 R CC 419 23S EC 971 CGAGAGGGAAACAACCC 23S EC 1059 21 992 F AGACC 64 1077 R TGGCTGCTTCTAAGCCAAC 400 AB MLST 11- AB MLST-11 01F007_120 TCGTGCCCGCAATTTGC 0IF007_1266 TAATGCCGGGTAGTGCAAT 1158 2 1225 F ATAAAGC 65 1296 R CCATTCTTCTAG 420 AB MLST 11- ABMLST-11 01F007 120 TCGTGCCCGCAATTTGC 0IF007_1299 1159 2 1225~F ATAAAGC 65 1316 R TGCACCTGCGGTCGAGCG 421

ABMLST

11- ABMLST-11 OIF007_123 TTGTAGCACAGCAAGGC OIF007 1335 TGCCATCCATAATCACGCC 1160 4 1264 F AAATTTCCTGAAAC 66 1362 R ATACTGACG 422

ABMLST

11- AB MLST-11 OIF007_132 TAGGTTTACGTCAGTAT 0I007 1422 TGCCAGTTTCCACATTTCA 1161 7 1356 F GGCGTGATTATGG 67 1448 5 CGTTCGTG 423

AB_MLST

11- AB_MLST-11 OIF007_134 TCGTGATTATGGATGGC OIF007_1470 TCGCTTGAGTGTAGTCATG 1162 5 1369 F AACGTGAA 68 1494 R ATTGCG 424

ASMLST

11- ABMLST-11 01F007_135 TTATGGATGGCAACGTG OIF007_1470 TCGCTTGAGTGTAGTCATG 1163 1 1375 F AAACGCGT 69 1494 R ATTGCG 424 AB MLST 11- ABMLST-11 OIF007 138 TCTTTGCCATTGAAGAT OIF007_1470 TCGCTTGAGTGTAGTCATG 1164 7 1412 F GACTTAAGC 70 1494 R ATTGCG 424

ABMLST

11- ABMLST-11 OIF007 154 TACTAGCGGTAAGCTTA OIF007 1656 TGAGTCGGGTTCACTTTAC 1165 2 1569 F AACAAGATTGC 71 1680 R CTGGCA 425 AB MLST 11- AB MLST-11 0IF007_156 TTGCCAATGATATTCGT OIF007_1656 TGAGTCGGGTTCACTTTAC 1166 6 1593 F TGGTTAGCAAG 72 1680 R CTGGCA 425

ABMLST

11- AB MLST-11 0IF007_161 TCGGCGAAATCCGTATT 01F007_1731 TACCGGAAGCACCAGCGAC 1167 1 1638 F CCTGAAAATGA 73 1757 R ATTAATAG 427

ABMLST

11- ABMLST-11 OIF007_172 TACCACTATTAATGTCG OIF007_1790 TGCAACTGAATAGATTGCA 1168 6 1752 F CTGGTGCTTC 74 1821 R GTAAGTTATAAGC 428

ABMLST

11- TTATAACTTACTGCAAT ABMLST-11 OIF007_179 CTATTCAGTTGCTTGGT OIF007_1876 TGAATTATGCAAGAAGTGA 1169 2 1826 F G 75 1909 R TCAATTTTCTCACGA 429

ABMLST

11- TTATAACTTACTGCAAT ABMLST-11 OIF007_179 CTATTCAGTTGCTTGGT OIF007_1895 TGCCGTAACTAACATAAGA 1170 2 1826 F G 75 1927 R GAATTATGCAAGAA 430

ABMLST

11- ABMLST-11 0IF007 185 TATTGTTTCAAATGTAC OIF007 291 TCACAGGTTCTACTTCATC 1152 214 F AAGGTGAAGTGCG 76 324 R~ AATAATTTCCATTGC 432

ABMLST

11- ABMLST-11 0IF007_197 TGGTTATGTACCAAATA OIF007_2097 TGACGGCATCGATACCACC 1171 0 2002 F CTTTGTCTGAAGATGG 77 2118 R GTC 431

AB_MLST

11- ABMLST-11 OIF007 206 TGAAGTGCGTGATGATA OIF007 318 TCCGCCAAAAACTCCCCTT 1154 239 F TCGATGCACTTGATGTA 78 344 R TTCACAGG 433 -28 AB MLST l T ABMLST-11 0IF007_260 TGGAACGTTATCAGGTG 01F007_364 TTGCAATCGACATATCCAT 153 289 F CCCCAAAAATTCG 79 393 R TTCACCATGCC 434 AB MLST 11- ABMLST-11 0IF007_522 TCGGTTTAGTAAAAGAA OIF007_587_ TTCTGCTTGAGGAATAGTG 1155 552 F CGTATTGCTCAACC 80 610 R CGTGG AB MLST 11- ASMLST-11 OIF007 547 TCAACCTGACTGCGTGA OIF007 656 TACGTTCTACGATTTCTTC 1156 571 F ' ATGGTTGT 81 686 R ATCAGGTACATC 436

ABMLST

11- ABMLST-11 OIF007_601 TCAAGCAGAAGCTTTGG OIF007710 TACAACGTGATAAACACGA 1157 627 F AAGAAGAAGG 82 736 R CCAGAAGC 437 AB MLST 11- ABMLST-11 OIF007 62 TGAGATTGCTGAACATT OIF007_169 TTGTACATTTGAAACAATA 1151 91 F TAATGCTGATTGA 83 203 R TGCATGACATGTGAAT 426 ASD FRT 1 TTGCTTAAAGTTGGTTT ASD_FRT_86_ TGAGATGTCGAAAAAAACG 1100 29 F TATTGGTTGGCG 84 116 R TTGGCAAAATAC 439 ASD FRT 43 TCAGTTTTAATGTCTCG ASD_FRT_129 TCCATATTGTTGCATAAAA 1101 76 F TATGATCGAATCAAAAG 85 156 R CCTGTTGGC 438 ASPS EC 40 GCACAACCTGCGGCTGC ASPSEC_521 291 5 422 F G 86 538 R ACGGCACGAGGTAGTCGC 440 BONTA X520 66_450_473 TCTAGTAATAATAGGAC BONTAX5206 TAACCATTTCGCGTAAGAT 485 F CCTCAGC 87 6 517 539 R TCAA 441 BONTA X520 T*U'*CAGTAATAATAG BONTA_X5206 66 450 473 GA***U**C**U*AG 6_517_539P TAACCA*C*C*Ca*4aGC 486 P F C 87 R GTAAGA*C'*C*UAA 441 BONTA X520 66_538_552 BONTAX5206 481 F TATGGCTCTACTCAA 88 6 647 660 R TGTTACTGCTGGAT 443 BONTA X520 BONTA X5206 66 538_552 TA*C'GGC*C**U*C*A \ 6_647_660P_ TG*C'*CA*U**CaG*Ua*C 482 P F *Ua88a*UaAA 8 R GGAT 443 BONTAX520 66_591_620 TGAGTCACTTGAAGTTG BONTA_X5206 TCATGTGCTAATGTTACTG 487 F ATACAAATCCTCT 89 6 644 671 R CTGGATCTG 442 BONTA X520 66 701 720 GAATAGCAATTAATCCA BONTAx5206 483 F~ ~ AAT 90 6 759 775 R TTACTTCTAACCCACTC 444 BONTA X520 BONTAX5206 66_701_720 GAA*C*AG*U*AA*Ca*C 6_759_775P_ TTA*Ua*C U**C*CAAA* 484 P F "AA*Ca*ga*gAAAT 90 R U.*U*A*U**CC 444 CAF1_AF053 CAF1_AF0539 947 33407 TCAGTTCCGTTATCGCC 47_33494_33 TGCGGGCTGGTTCAACAAG 774 33430 F- ATTGCAT 91 514 R AG 445 CAF1_AF053 CAFIAF0539 947_33435 TGGAACTATTGCAACTG 47_33499_33 776 33457 F CTAATG 92 517 R TGATGCGGGCTGGTTCAAC 446 CAF1_AF053 CAF1_AF0539 947 33515 TCACTCTTACATATAAG 47_33595_33 TCCTGTTTTATAGCCGCCA . 775 33541 F GAAGGCGCTC 93 621 R AGAGTAAG 447 CAFI AF053 CAF1_AF0539 947 3687 TCAGGATGGAAATAACC 47_33755_33 TCAAGGTTCTCACCGTTTA 777 33716 F ACCAATTCACTAC 94 782 R CCTTAGGAG 448 CAPCBA 10 GTTATTTAGCACTCGTT CAPCBA_180 TGAATCTTGAAACACCATA 22 4 131 F TTTAATCAGCC 95 205 R CGTAACG 449 CAPC BA 11 ACTCGTTTTTAATCAGC CAPCBA_185 TGAATCTTGAAACACCATA 23 4 133 F CCG 96 205 R CG 450 CAPCBA_27 GATTATTGTTATCCTGT CAPCBA_349 GTAACCCTTGTCTTTGAAT 24 4 303 F TATGCCATTTGAG 97 376 R TGTATTTGC 451 CAPCBA_27 4 303 TOD TGATTATTGTTATCCTG CAPCBA_349 TGTAACCCTTGTCTTTGAA 350 i ~ TTATGCCATTTGAG 98 376 TMOD R TTGTATTTGC . 452 CAPC BA 27 TTATTGTTATCCTGTTA CAPCBA 358 GGTAACCCTTGTCTTTGAA 25 6 296 F~ TGCC 99 377 R T 453 CAPC BA 28 GTTATCCTGTTATGCCA CAPCBA_361 26 1 301 F~ TTTG 100 378 R TGGTAACCCTTGTCTTTG 454 CAPCBA_31 CCGTGGTATTGGAGTTA CAPCBA_361 27 5 334 F TTG 101 378 R TGGTAACCCTTGTCTTTG 454 1053 CJST CJ 10 TTGAGGGTATGCACCGT 102 CJST CJ 116 TCCCCTCATGTTTAAATGA 456 -29 80 1110 F CTTTTTGATTCTTT 6 1198 R TCAGGATAAAAAGC CJST CJ 12 AGTTATAAACACGGCTT CJST_CJ 134 TCGGTTTAAGCTCTACATG 1063 68 1299 F TCCTATGGCTTATCC 103 9 1379 R ATCGTAAGGATA 457 CJST CJ 12 TGGCTTATCCAAATTTA CJSTCJ_140 TTTGCTCATGATCTGCATG 1050 90 1320 F GATCGTGGTTTTAC 104 6 1433 R AAGCATAAA 458 CJST CJ 16 TTATCGTTTGTGGAGCT CJSTCJ_172 TGCAATGTGTGCTATGTCA 1058 43 1670 F AGTGCTTATGC 105 4 1752 R GCAAAAAGAT 459 CJST CJ 16 TGCTCGAGTGATTGACT CJST CJ_17 TGAGCGTGTGGAAAAGGAC 1045 68 1700 F TTGCTAAATTTAGAGA 106 4 1799 R TTGGATG 460 CJST CJ 16 TGATTTTGCTAAATTTA CJST-CJ_179 TATGTGTAGTTGAGCTTAC 1064 80 1713 F GAGAAATTGCGGATGAA 107 5 1822 R TACATGAGC 461 CJST-CJ_18 TCCCAATTAATTCTGCC CJST_CJ_198 TGGTTCTTACTTGCTTTGC 1056 80 1910 F ATTTTTCCAGGTAT 108 1 2011 R ATAAACTTTCCA 462 CJST CJ20 TCCCGGACTTAATATCA CJST_CJ_214 TCGATCCGCATCACCATCA 1054 60 2090 F ATGAAAATTGTGGA 109 8 2174 R AAAGCAAA 463 CJSTCJ_21 TGCGGATCGTTTGGTGG CJST_CJ_224 TCCACACTGGATTGTAATT 1059 65 2194 F TTGTAGATGAAAA 110 7 2278 R TACCTTGTTCTTT 464 CJSTCJ_21 TCGTTTGGTGGTGGTAG CJSTCJ_228 TCTCTTTCAAAGCACCATT 1046 71 2197 F ATGAAAAAGG 111 3 2313 R GCTCATTATAGT 465 CJST CJ 21 TAGATGAAAAGGGCGAA CJSTCJ_228 TGAATTCTTTCAAAGCACC 1057 85 2212 F GTGGCTAATGG 112 3, 2316 R ATTGCTCATTATAGT 466 CJST Ca 26 TGCCTAGAAGATCTTAA CJSTCJ_275 TTGCTGCCATAGCAAAGCC 1049 36 2668 F AAATTTCCGCCAACTT 113 3 2777 R TACAGC 467 CJSTCJ26 TCCCCAGGACACCCTGA CJST_Ca_276 TGTGCTTTTTTTGCTGCCA 1062 78 2703 F AATTTCAAC 114 0 2787 R TAGCAAAGC 468 CJST CJ 28 TGGCATTTCTTATGAAG CJSTCJ_296 TGCTTCAAAACGCATTTTT 1065 57 2887 F CTTGTTCTTTAGCA 115 5 2998 R ACATTTTCGTTAAAG 469 CJST CJ 28 TGAAGCTTGTTCTTTAG CJST_CJ 297 TCCTCCTTGTGCCTCAAAA 1055 69 2895 F CAGGACTTCA 116 9 3007 R9 CGCATTTTTA 470 CJSTCJ_32 TTTGATTTTACGCCGTC CJST_CJ_335 TCAAAGAACCCGCACCTAA 1051 67 3293 F CTCCAGGTCG 117 6 3385 R TTCATCATTTA 471 CJSTCJ_36 TCCTGTTATCCCTGAAG CJSTCJ_443 TACAACTGGTTCAAAAACA 1061 0 393 F TAGTTAATCAAGTTTGT 118 477 R TTAAGCTGTAATTGTC 473 TCCTGTTATCCCTGAAG CJST CJ 36 TAGTTAATCAAGTTTGT CJST CJ 442 TCAACTGGTTCAAAAACAT 1048 0 394 F T 119 476 R TAAGTTGTAATTGTCC 472 TAGGCGAAGATATACAA CJST CJ 5 AGAGTATTAGAAGCTAG CJST CJ 104 TCCCTTATTTTTCTTTCTA 1052 39 F A 120 137 R CTACCTTCGGATAAT 455 CJST CJ 58 TCCAGGACAAATGTATG CJSTCJ 663 TTCATTTTCTGGTCCAAAG 1047 4 616 F~ AAAAATGTCCAAGAAG 121 692 a TAAGCAGTATC 474 CJST CJ 59 TGAAAAATGTCCAAGAA CJST_CJ_711 TCCCGAACAATGAGTTGTA 1060 9 632 F~ GCATAGCAAAAAAAGCA 122 743 R TCAACTATTTTTAC 475 CTXAVBC_1 TCTTATGCCAAGAGGAC CTXAVBC_19 TGCCTAACAAATCCCGTCT 1096 17 142 F AGAGTGAGT 123 4 218 R GAGTTC 476 CTXA VBC 3 TGTATTAGGGGCATACA CTXA_VBC_44 TGTCATCAAGCACCCCAAA 1097 51 377 F- GTCCTCATCC 124 1 466 R ATGAACT 477 CYA BA 105 GAAAGAGTTCGGATTGG CYABA_1112 28 5 1072 F G 125 1130 R TGTTGACCATGCTTCTTAG 479 CYABA_134 ACAACGAAGTACAATAC CYABA_1426 CTTCTACATTTTTAGCCAT 277 9 1370 F AAGAC 126 1447 R CAC 480 CYA BA 135 CGAAGTACAATACAAGA CYABA 1448 TGTTAACGGCTTCAAGACC 30 3 1379 F CAAAAGAAGG 127 1467 R C 482 CYA BA 135 CYABA 1448 3_1379-TMO TCGAAGTACAATACAAG _1467_TMOD_ TTGTTAACGGCTTCAAGAC 351 D F ACAAAAGAAGG 128 R CC 483 CYABA_135 ACAATACAAGACAAAAG CYABA_1447 31 9 1379 F - AAGG 129 1461 R CGGCTTCAAGACCCC 481 CYA BA 914 CAGGTTTAGTACCAGAA CYABA 999 ACCACTTTTAATAAGGTTT 32 937 F CATGCAG t 130 1026 R. GTAGCTAAC 484 CYA BA 916 GGTTTAGTACCAGAACA CYABA_1003 CCACTTTTAATAAGGTTTG 33 935 F TGC 131 1025 R TAGC 478 DNAKEC_42 CGGCGTACTTCAACGAC DNAKEC_503 CGCGGTCGGCTCGTTGATG 115 8 449 F AGCCA 132 522 R A 485 GALE FRT_1 TTATCAGCTAGACCTTT GALE_FRT_24 TCACCTACAGCTTTAAAGC 1102 68 199 F TAGGTAAAGCTAAGC 133 1 269 R CAGCAAAATG 486 GALEFRT 3 TCCAAGGTACACTAAAC GALE_FRT_39 TCTTCTGTAAAGGGTGGTT 1104 08 339 F TTACTTGAGCTAATG 134 0 422 R TATTATTCATCCCA 487 GALE FRT_8 TCAAAAAGCCCTAGGTA GALEFRT_90 TAGCCTTGGCAACATCAGC 1103 34 865 F AAGAGATTCCATATC 135 1 925 R AAAACT 488 GLTARKP_1 TCCGTTCTTACAAATAG GLTARKP_11 TTGGCGACGGTATACCCAT 1092 023 1055 F CAATAGAACTTGAAGC 136 29 1156 R AGCTTTATA 489 GLTA RKP 1 043 1072_2 TGGAGCTTGAAGCTATC GLTA_RKP 11 TGAACATTTGCGACGGTAT 1093 F GCTCTTAAAGATG 137 38 1162 Ri ACCCAT 490 -30 GLTA RKP 1 043_1072_3 TGGAACTTGAAGCTCTC GLTARKP_11 TGTGAACATTTGCGACGGT 1094 F GCTCTTAAAGATG 138 38 1164 R ATACCCAT 492 GLTA_RKP_1 TGGGACTTGAAGCTATC GLTARKP_11 TGAACATTTGCGACGGTAT 1090 043 1072 F GCTCTTAAAGATG 139 38 1162 R ACCCAT 491 GLTARKP_4 TCTTCTCATCCTATGGC GLTARKP_49 TGGTGGGTATCTTAGCAAT 1091 00 428 F TATTATGCTTGC 140 9 529 R CATTCTAATAGC 493 GLTA RKP_4 TCTTCTCATCCTATGGC GLTA_RKP_50 TGCGATGGTAGGTATCTTA 1095 00 428 F TATTATGCTTGC 140 5 534 R GCAATCATTCT 494 GROLEC 21 GGTGAAAGAAGTTGCCT GROLEC_328 TTCAGGTCCATCGGGTTCA 224 9 242 F CTAAAGC 141 350 R TGCC 496 GROLEC_49 ATGGACAAGGTTGGCAA GROLEC_577 TAGCCGCGGTCGAATTGCA 280 6 518 F GGAAGG 142 596 R T 498 GROLEC51 AAGGAAGGCGTGATCAC GROLEC_571 CCGCGGTCGAATTGCATGC 281 1 536 F CGTTGAAGA 143 593 R CTTC 497 GROL EC_94 TGGAAGATCTGGGTCAG GROL_TC_103 CAATCTGCTGACGGATCTG 220 1 959 F GC 144 9 1060 R AGC 495 GYRA AF1005 GYRAAF100 TCTGCCCGTGTCGTTGG 57_119_142 TCGAACCGAAGTTACCCTG 924 557 4 23 F TGA 145 R ACCAT 499 GYRA AF100 GYRAAF1005 557_270 94 TCCATTGTTCGTATGGC 57_178 201 TGCCAGCTTAGTCATACGG 925 F TCAAGACT 146 R ACTTC 500 GYRB_AB008 GYRB_AB0087 700_19_40 TCAGGTGGCTTACACGG 00_111_140_ TATTGCGGATCACCATGAT 926 F CGTAG 147 R GATATTCTTGC 501 GYRBAB008 GYRBAB0087 700_265_29 TCTTTCTTGAATGCTGG 00_369 395_ TCGTTGAGATGGTTTTTAC 927 2 F TGTACGTATCG 148 R CTTCGTTG 502 GYRB AB008 GYRBAB0087 '700_368_39 TCAACGAAGGTAAAAAC 00_466_494_ TTTGTGAAACAGCGAACAT 928 4 F CATCTCAACG 149 R TTTCTTGGTA 503 GYRB_ABOO8 GYRBAB0087 700_477_50 TGTTCGCTGTTTCACAA 00_611_632_ TCACGCGCATCATCACCAG 929 4 F ACAACATTCCA 150 R TCA 504 GYRBABOO GYRB_AB0087 700_760_78 TACTTACTTGAGAATCC 00_862_88_ TCCTGCAATATCTAATGCA 949 7 F ACAAGCTGCAA 151 2 R CTCTTACG 505 GYRBABOO8 GYRB AB0087 700_760_78 TACTTACTTGAGAATCC 00_662 888 ACCTGCAATATCTAATGCA 930 7 F ACAAGCTGCAA 151 R. CTCTTACG 506 HFLBEC_10 TGGCGAACCTGGTGAAC HFLB _EC_114 CTTTCGCTTTCTCGAACTC 222 82 1102 F GAAGC 152 4 1168 R AACCAT 507 HOPBCJ_11 TAGTTGCTCAAACAGCT HUPBCJ_157 TCCCTAATAGTAGAAATAA 1128 3 134 F GGGCT 153 188 R CTGCATCAGTAGC 509 HUPBCJ_76 TCCCGGAGCTTTTATGA HUPBCJ 114 TAGCCCAGCTGTTTGAGCA 1130 102 F CTAAAGCAGAT 154 135 R ACT 508 HUPBCJ 76 TCCCGGAGCTTTTATGA HUPBCJ_157 TCCCTAATAGTAGAAATAA 1129 102 F CTAAAGCAGAT 154 188 R CTGCATCAGTAGC 510 ICDCXB_17 TCGCCGTGGAAAAATCC ICDCXB_224 TAGCCTTTTCTCCGGCGTA 1079 6 198 F TACGCT 155 247 R GATCT 512 ICDCXB_92 TTCCTGACCGACCCATT ICDCXB_172 TAGGATTTTTCCACGGCGG 1078 120 F ATTCCCTTTATC 156 194 R CATC 510 ICD CXB_93 TCCTGACCGACCCATTA ICDCXB_172 TAGGATTTTTCCACGGCGG 1077 120 F TTCCCTTTATC 157 194 R CATC 511 INFBEC_11 GTCGTGAAAACGAGCTG INFBEC_117 221 03 1124 F GAAGA 158 4 1191 ft CATGATGGTCACAACCGG 513 INFBEC_13 TGCGTTTACCGCAATGC INFBEC_141 964 47 1367 F GTGC 159 4 1432 R TCGGCATCACGCCGTCGTC 514 INFB_EC_13 TGCTCGTGGTGCACAAG INFBEC_143 TGCTGCTTTCGCATGGTTA 34 65 1393 F TAACGGATATTA 160 9 1467 R ATTGCTTCAA 515 INFB EC 13 INFB EC_143 65 1393_TM TTGCTCGTGGTGCACAA 9_1467_TMOD TTGCTGCTTTCGCATGGTT 352 OD F GTAACGGATATTA 161 Rt AATTGCTTCAA 516 INFB_EC_19 CGTCAGGGTAAATTCCG INFBEC_203 AACTTCGCCTTCGGTCATG 223 69 1994 F TGAAGTTAA 162 8 2058 R TT 517 INV U22457 1558_1581 TGGTAACAGAGCCTTAT INV 022457 TTGCGTTGCAGATTATCTT 781 F AGGCGCA 163 1619 1643 R TACCAA 518 INVU22457 TGGCTCCTTGGTATGAC INV U22457_ TGTTAAGTGTGTTGCGGCT 778 515 539 F TCTGCTTC 164 571 598 R GTCTTTATT 519 INVU22457 TGCTGAGGCCTGGACCG INV_022457_ TCACGCGACGAGTGCCATC 779 699 724 F ATTATTTAC 165 753 776 R CATTG 520 INVU22457 TTATTTACCTGCACTCC INV 022457_ TGACCCAAAGCTGAAAGCT 780 834 858 F CACAACTG 166 942 966 R TTACTG 521 -31 IPAH SGF 1 TCCTTGACCGCCTTTCC IPAHSGF 17 TTTTCCAGCCATGCAGCGA 1106 13 134 F GATAC 167 2 191 R C 522 IPAHSGF_2 TGAGGACCGTGTCGCGC IPAH SGF 30 TCCTTCTGATGCCTGATG 1105 58 277 F TCA 168 1 327 R ACCAGGAG 523 IPAH SGF 4 TCAGACCATGCTCGCAG IPAHSGF_52 1107 62 486 F~ AGAAACTT 169 2 540 R TGTCACTCCCGACACGCCA 524 IS1111A NC IS1111ANCO 002971_686 TCAGTATGTATCCACCG 02971_6928 TAAACGTCCGATACCAATG 1080 6 6891 F TAGCCAGTC 170 6954 R GTTCGCTC 525 IS1111A NC IS1111ANCO 002971 745 TGGGTGACATTCATCAA 02971 7529 TCAACAACACCTCCTTATT 1081 6 7483 F TTTCATCGTTC 171 7554 Ri CCCACTC 526 LEF BA 103 LEFBA_1119 35 3 1052 F TCAAGAAGAAAAAGAGC 172 1135 R GAATATCAATTTGTAGC 527 LEF BA_103 CAAGAAGAAAAAGAGCT LEFBA_1119 AGATAAAGAATCACGAATA 36 6 1066 F TCTAAAAAGAATAC 173 1149 R TCAATTTGTAGC 528 LEFBA_756 AGCTTTTGCATATTATA LEFBA 843_ TCTTCCAAGGATAGATTTA 37 781 F TCGAGCCAC 174 872 R TTTCTTGTTCG 530 LEFBA_756 781_TMOD_ TAGCTTTTGCATATTAT LEF_BA_843_ TTCTTCCAAGGATAGATTT 353 F ATCGAGCCAC 175 872 TMOD R ATTTCTTGTTCG 531 LEFBA_758 CTTTTGCATATTATATC LEFBA_843_ AGGATAGATTTATTTCTTG 38 778 F GAGC 176 865 R TTCG 529 LEF BA 795 TTTACAGCTTTATGCAC LEFBA 883 39 813 F CG 177 900 Rt TCTTGACAGCATCCGTTG 532 LEFBA_883 LEFBA_939_ CAGATAAAGAATCGCTCCA 40 899 F CAACGGATGCTGGCAAG 178 958 R G 533 LL NC00314 LL NCO03143 3 366996 TGTAGCCGCTAAGCACT _2367073_23 TCTCATCCCGATATTACCG 782 2367019 F ACCATCC 179 67097 R CCATGA 534 LL NC00314 LLNC003143 3 2367172 TGGACGGCATCACGATT 2367249_23 TGGCAACAGCTCAACACCT 783 2367194 F CTCTAC 180 67271 R TTGG 535 MECA Y1405 MECAY14051 1 3645_367 TGAAGTAGAAATGACTG 3690 3719 TGATCCTGAATGTTTATAT 878 OF AACGTCCGA 181 R CTTTAACGCCT 536 MECA Y1405 MECAY14051 1 3774 380 TAAAACAAACTACGGTA 3828_3854_ TCCCAATCTAACTTCCACA 877 2 F ACATTGATCGCA 182 R TACCATCT 537 MECAY1405 MECA_Y14051 1_4507_453 TCAGGTACTGCTATCCA 4555_4581_ TGGATAGACGTCATATGAA 879 0 F CCCTCAA '183 R _ GGTGTGCT 538 MECAY1405 MECA_Y14051 14510_453 TGTACTGCTATCCACCC 4586_4610 TATTCTTCGTTACTCATGC 880 0 F TCAA 184 R CATACA 539 MECA Y1405 MECAY14051 1_4520_453 4590 4600P 882 OP F TUOAUaWUgCgUAA 185 R CAUC*U*AC*GUUA 540 MECAJ 1405 MECAY14051 1_4520_453 4600_4610P 883 OP F TU*AU*U"U'C*UCAA 185 R ~ C*ACaC'U"C"CaaGC*T 541 MECA Y1405 MECAY14051 1_4669_469 TCACCAGGTTCAACTCA 4765_4793 TAACCACCCCAAGATTTAT 881 8 F AAAAATATTAACA 186 R CTTTTTGCCA 542 MECIA Y140 MECIA Y1405 51 3315 33 TTACACATATCGTGAGC 1_3367_3393 TGTGATATGGAGGTGTAGA 876 41 F ~ AATGAACTGA 187 R AGGTGTTA 543 OMPAAY485 OMPAAY4852 227_272_30 TTACTCCATTATTGCTT 27_364388 GAGCTGCGCCAACGAATAA 914 1 F GGTTACACTTTCC 188 R ATCGTC 544 OMPA AY485 OMPAAY4852 227 311 33 TACACAACAATGGCGGT 27_424 453 TACGTCGCCTTTAACTTGG 916 5 F AAAGATGG 189 R TTATATTCAGC 545 OMPAAY485 OMPAAY4852 227_379_40 TGCGCAGCTCTTGGTAT 27_492519 TGCCGTAACATAGAAGTTA 915 1 F CGAGTT 190 R CCGTTGATT 546 OMPA AY485 OMPAAY4852 227 415 44 TGCCTCGAAGCTGAATA 27514_546 TCGGGCGTAGTTTTTAGTA 917 1 F , TAACCAAGTT 191 R ATTAAATCAGAAGT 547 OMPAAY485 OMPAAY4852 227_494_52 TCAACGGTAACTTCTAT 27_569 596 TCGTCGTATTTATAGTGAC 918 0 F GTTACTTCTG 192 R CAGCACCTA 548 OMPAAY485 OMPAAY4852 227 551 57 TCAAGCCGTACGTATTA 27_658_680 TTTAAGCGCCAGAAAGCAC 919 7 F TTAGGTGCTG 193 R CAAC 550 -32 OMPA AY485 OMPAAY4852 227 555 58 TCCGTACGTATTATTAG 27_635_662_ TCAACACCAGCGTTACCTA 920 1 F GTGCTGGTCA 194 R AAGTACCTT 549 OMPA-AY485 OMPA AY4852 227556_58 TCGTACGTATTATTAGG 27_659_683 TCGTTTAAGCGCCAGAAAG 921 3 F TGCTGGTCACT 195 R CACCAA 551 OMPAAY485 OMPAAY4852 227 657 67 TGTTGGTGCTTTCTGGC 27_739_765 TAAGCCAGCAAGAGCTGTA 922 9 F GCTTAA 196 R TAGTTCCA 552 OMPA AY485 OMPAAY4852 227_660_68 TGGTGCTTTCTGGCGCT 27_786 807 TACAGGAGCAGCAGGCTTC 923 3 F TAAACGA 197 R AAG 553 OMPB RKP_ 1 TCTACTGATTTTGGTAA OMPB_RKP_12 TAGCAGCAAAAGTTATCAC 1088 192 1221 F TCTTGCAGCACAG 198 88 1315 R ACCTGCAGT 554 OMPB RKP_3 TGCAAGTGGTACTTCAA OMPBRKP_35 TGGTTGTAGTTCCTGTAGT 1089 417 3440 F CATGGGG 199 20 3550 R TGTTGCATTAAC 555 OMPB RKP_8 TTACAGGAAGTTTAGGT OMPBRKP_97 TCCTGCAGCTCTACCTGCT 1087 60 890 F GGTAATCTAAAAGG 200 2 996 R CCATTA 556 PAG BA 122 CAGAATCAAGTTCCCAG PAGBA 190 CCTGTAGTAGAAGAGGTAA 41 14i F GGG 201 2097R C 558 PAGBA_123 AGAATCAAGTTCCCAGG PAGBA_187_ CCCTGTAGTAGAAGAGGTA 42 145 F GGTTAC 202 210 R ACCAC 557 PAGBA_269 AATCTGCTATTTGGTCA PAGBA_326 43 287 F GG 203 344 R TGATTATCAGCGGAAGTAG 559 PAG Ba_655 GAAGGATATACGGTTGA PAGBA 755 44 675 F TGTC 204 772 R CCGTGCTCCATTTTTCAG 560 PAGBA_753 TCCTGAAAAATGGAGCA PAGBA 849_ TCGGATAAGCTGCCACAAG 45 772 F CGG 205 868 R G 561 PAG BA 763 TGGAGCACGGCTTCTGA PAGBA 849 TCGGATAAGCTGCCACAAG 46 781 F TC 206 868 R G 562 PARC X9581 9 123 141 GGCTCAGCCATTTAGTT PARC X95819 TCGCTCAGCAATAATTCAC 912 F ACCGCTAT 207 232 260 R TATAAGCCGA 566 PARC X9581 TCAGCGCGTACAGTGGG PARCX95819 TTCCCCTGACCTTCGATTA 913 9 43 63 F TGAT 208 143 170 R AAGGATAGC 563 PARC X9581 TGGTGACTCGGCATGTT PARCX95819 GGTATAACGCATCGCAGCA 911 9 87 110 F ATGAAGC 209 192 219 R AAAGATTTA 564 PARC X9581 TGGTGACTCGGCATGTT PARCX95819 TTCGGTATAACGCATCGCA 910 9 87 110 F ATGAAGC 209 201 222 R GCA 565 PLA AF0539 PLAAF05394 45 7186_72 TTATACCGGAAACTTCC 5_7257_7280 TAATGCGATACTGGCCTGC 773 11 F CGAAAGGAG 210 R AAGTC 567 PLA AF0539 PLAAF05394 45 5377 74 TGACATCCGGCTCACGT 5_7434_7462 TGTAAATTCCGCAAAGACT 770 02 F TATTATGGT 211 R TTGGCATTAG 568 PLA AF0539 PLAAF05394 45_7382 74 TCCGGCTCACGTTATTA 5_7482_7502 TGGTCTGAGTACCTCCTTT 771 04 F TGGTAC 212 R GC 569 PLA AF0539 PLA AF05394 45_7481_75 TGCAAAGGAGGTACTCA 5_7539_7562 TATTGGAAATACCGGCAGC 772 03 F GACCAT 213 R ATCTC 570 RECAAF251 RECAAF2514 469_169_19 TGACATGCTTGTCCGTT 69_277_300 TGGCTCATAAGACGCGCTT 909 0 F CAGGC 214 R GTAGA 572 RECA AF251 RECAAF2514 46943 68 TGGTACATGTGCCTTCA 69 140 163 TTCAAGTGCTTGCTCACCA 908 F TTGATGCTG 215 R TTGTC 571 RNASEP BDP TGGCACGGCCATCTCCG RNASEPBDP_ TCGTTTCACCCTGTCATGC 1072 574 592 F TG 216 616 635 R CG 573 RNASEP BRM TGCGGGTAGGGAGCTTG RNASEPBKM_ TCCGATAAGCCGGATTCTG 1070 580 599 F AGC 217 665 686 R TGC 574 RNASEP BKM TCCTAGAGGAATGGCTG RNASEPBKM_ TGCCGATAAGCCGGATTCT 1071 616 637 F CCACG 218 665 687 R GTGC 575 RNASEP BRM TACCCCAGGGAAAGTGC RNASEPBRM_ TCTCTTACCCCACCCTTTC 1112 325 347 F CACAGA 219 402 428 R ACCCTTAC 576 RNASEP BRM TAAACCCCATCGGGAGC RNASEPBRM_ TGCCTCGTGCAACCCACCC 1172 461 488 F AAGACCGAATA 220 542 561 2 R G 577 RNASEP BRM TAAACCCCATCGGGAGC RNASEP BRM TGCCTCGCGCAACCTACCC 1111 461 488 F AAGACCGAATA 220 542 561 R G 578 RNASEPBS_ GAGGAAAGTCCATGCTC RNASEPBS_3 GTAAGCCATGTTTTGTTCC 258 43 61 F GC 221 63 384 R ATC 579 RNASEP BS_ GAGGAAAGTCCATGCTC RNASEP_BS_3 GTAAGCCATGTTTTGTTCC 259 43 61 F GC 221 63 384 R ATC 578 RNASEPBS_ GAGGAAAGTCCATGCTC RNASEPEC_3 258 43 61 F GC 221 45 362 R ATAAGCCGGGTTCTGTCG 581 -33 RNASEP BS GAGGAAAGTCCATGCTC RNASEPSA_3 ATAAGCCATGTTCTGTTCC 258 43 61 F GC 221 58379R ATC 584 RNASEPN CL TAAGGATAGTGCAACAG RNASEPCLB_ TTTACCTCGCCTTTCCACC 1076 459 487 F AGATATACCGCC 222 498 522 R CTTACC 579 RNASEP CLB TAAGGATAGTGCAACAG RNASEP_CLB_ TGCTCTTACCTCACCGTTC 1075 459 487 F AGATATACCGCC 222 498 526 R CACCCTTACC 580 RNASEPEC RNASEPBS_3 GTAAGCCATGTTTTGTTCC 258 61 77 F GAGGAAAGTCCGGGCTC 223 63 384 R ATC 578 PNASEP EC RNASEP_EC_3 258 61 77 F GAGGAAAGTCCGGGCTC 223 45 362 R ATAAGCCGGGTTCTGTCG 581 RNASEP EC RNASEPEC_3 260 61 77 F GAGGAAAGTCCGGGCTC 223 45 362 R ATAAGCCGGGTTCTGTCG 581 RNASEPEC RNASEPSA_3 ATAAGCCATGTTCTGTTCC 258 61 77 F GAGGAAAGTCCGGGCTC 223 58 379 R ATC 584 RNASEP RKP TCTAAATGGTCGTGCAG RNASEPRKP_ TCTATAGAGTCCGGACTTT 1085 264 287 F TTGCGTG 224 295 321 R CCTCGTGA 582 RNASEP RKP TGGTAAGAGCGCACCGG RNASEPRKP TCAAGCGATCTACCCGCAT 1082 419 48 F TAAGTTGGTAACA 225 542 565 R TACAA 583 RNASEP RKP TAAGAGCGCACCGGTAA RNASEP_RKP_ TCAAGCGATCTACCCGCAT 1083 422 443 F GTTGG 226 542 565 R TACAA 583 RNASEP RKP TGCATACCGGTAAGTTG RNASEPRKP_ TCAAGCGATCTACCCGCAT 1086 426 448 F GCAACA 227 542 565 R TACAA 583 RNASEP RKP TCCACCAAGAGCAAGAT RNASEP RKP_ TCAAGCGATCTACCCGCAT 1084 466 491 F CAAATAGGC 228 542 565 R TACAA 583 RNASEPSA_ GAGGAAAGTCCATGCTC RNASEPBS_3 GTAAGCCATGTTTTGTTCC 258 31 49 F AC 229 63 384 R ATC 578 RNASEPSA_ GAGGAAAGTCCATGCTC RNASEPEC_3 258 31 49 F AC 229 45 362 R ATAAGCCGGGTTCTGTCG 581 RNASEP_SA_ GAGGAAAGTCCATGCTC RNASEP_SA 3 ATAAGCCATGTTCTGTTCC 258 31 49 F AC 229 58 379 R* ATC 584 RNASEP SA GAGGAAAGTCCATGCTC RNASEP SA 3 ATAAGCCATGTTCTGTTCC 262 31 49 F AC 229 58 379 R ATC 584 RNASEPVBC TCCGCGGAGTTGACTGG RNASEP VBC_ TGACTTTCCTCCCCCTTAT 1098 331 349 F GT 230 388 414 R CAGTCTCC 585 RPLB EC 65 GACCTACAGTAAGAGGT RPLBEC_739 TCCAAGTGCTGGTTTACCC 66 0 679 F~ TCTGTAATGAACC 231 762 R CATGG 591 RPLB EC 65 0_679_TMOD TGACCTACAGTAAGAGG RPLBEC 739 TTCCAAGTGCTGGTTTACC 356 F TTCTGTAATGAACC 232 762 TMOD R CCATGG 592 RPLB EC 66 TGTAATGAACCCTAATG RPLB_EC_735 CCAAGTGCTGGTTTACCCC 73 9 69W F ACCATCCACACGG 233 761 R ATGGAGTA 586 RPLB EC 67 TAATGAACCCTAATGAC RPLBEC 737 TCCAAGTGCTGGTTTACCC 74 1 700 F~ CATCCACACGGTG 234 762 R CATGGAG 590 RPLB EC 68 CATCCACACGGTGGTGG RPLB_EC 736 GTGCTGGTTTACCCCATGG 67 8 710 F TGAAGG 235 757 R AGT 587 RPLB EC 68 CATCCACACGGTGGTGG RPLBEC_743 TGTTTTGTATCCAAGTGCT 70 8 710 F~ TGAAGG 235 771 R GGTTTACCCC 593 RPLB EC 68 8 71_ TMOD TCATCCACACGGTGGTG RPLBEC_736 TGTGCTGGTTTACCCCATG 357 F GTGAAGG 236 757 TMOD R GAGT 588 RPLB EC 69 TCCACACGGTGGTGGTG RPLBEC_737 TGTGCTGGTTTACCCCATG 449 0 710 F AAGG 237 758 R GAG 589 RPOBEC_13 GACCACCTCGGCAACCG RPOBEC 143 113 36 1353 F T 238 *8 1455 R TTCGCTCTCGGCCTGGCC 594 RPOB EC 15 TCAGCTGTCGCAGTTCA RPOB' EC_163 TCGTCGCGGACTTCGAAGC 963 27 15497F TGGACC 239 0 1649 R C 595 RPOBEC_18 TATCGCTCAGGCGAACT RPOBEC_190 GCTGGATTCGCCTTTGCTA 72 45 1866 F CCAAC 240 9 1929 R CG 596 RPOBEC_18 RPOBEC_190 45_1866_TM TTATCGCTCAGGCGAAC 9_1929_TMOD TGCTGGATTCGCCTTTGCT 359 OD F TCCAAC 241 R ACG 597 RPOB EC 20 TCGTTCCTGGAACACGA RPOBEC_204 TTGACGTTGCATGTTCGAG 962 05 2027 F TGACGC 242 1 2064 R CCCAT 598 RPOBEC_37 TCAACAACCTCTTGGAG RPOBEC_383 TTTCTTGAAGAGTATGAGC 69 62 3790 F GTAAAGCTCAGT 243 6 3865 R TGCTCCGTAAG 600 RPOB EC 37 CTTGGAGGTAAGTCTCA RPOBEC_382 CGTATAAGCTGCACCATAA 111 75 303 F TTTTGGTGGGCA 244 9 3858 R GCTTGTAATGC 599 RPOB EC 37 TGGGCAGCGTTTCGGCG RPOBEC_386 TGTCCGACTTGACGGTTAG 940 98 321 F AAATGGA 245 2 3889 2 R CATTTCCTG 604 RPOB EC_37 TGGGCAGCGTTTCGGCG RPOBEC_386 TGTCCGACTTGACGGTCAG 939 98 3821 F AAATGGA 245 2 3889 R CATTTCCTG 605 RPOB-EC_37 GGGCAGCGTTTCGGCGA RPOB_EC 386 GTCCGACTTGACGGTCAAC 289 99 3821 F AATGGA 246 2 3888 R ATTTCCTG 602 RPOB EC 37 TGGGCAGCGTTTCGGCG RPOBEC_386 TGTCCGACTTGACGGTCAA 362 99 3821~TM AAATGGA 245 2 3888 TMOD CATTTCCTG 603 -34 OD F R RPOBEC_38 CAGCGTTTCGGCGAAAT RPOBEC_386 CGACTTGACGGTTAACATT 288 02 3821 F GGA 247 2 3885 R TCCTG 601 RPOCEC_10 18_1045 2 CAAAACTTATTAGGTAA RPOC EC 109 TCAAGCGCCATCTCTTTCG 48 F GCGTGTTGACT 248 5 1124 i R GTAATCCACAT 610 RPOCEC 10 CAAAACTTATTAGGTAA RPOC EC_109 TCAAGCGCCATTTCTTTTG 47 18 1045 F GCGTGTTGACT 248 5 1124 R GTAAACCACAT 611 RPOCEC_10 CGTGTTGACTATTCGGG RPOCEC_109 ATTCAAGAGCCATTTCTTT 68 36 1060 F GCGTTCAG 249 7 1126 fR TGGTAAACCAC 612 RPOCEC_11 TAAGAAGCCGGAAACCA RPOC_EC_213 GGCGCTTGTACTTACCGCA 49 4 140 F TCAACTACCG 250 232 R C 617 RPOCEC 12 ACCCAGTGCTGCTGAAC RPOCEC_129 GTTCAAATGCCTGGATACC 227 56 1277 F CGTGC 251 5 1315 R CA 613 RPOCEC_13 CGCCGACTTCGACGGTG RPOC_EC_143 292 74 1393 F ACC 252 7 1455 R GAGCATCAGCGTGCGTGCT 614 RPOC_EC_13 RPOCEC_143 74_1393_TM TCGCCGACTTCGACGGT 7_1455_TMOD TGAGCATCAGCGTGCGTGC 364 ODF GACC 253 R T 615 RPOC EC 15 TGGCCCGAAAGAAGCTG RPOCEC_162 ACGCGGGCATGCAGAGATG 229 84 1604 F AGCG 254 3 1643 R CC 616 RPOCEC_21 TCAGGAGTCGTTCAACT RPOCEC_222 TTACGCCATCAGGCCACGC 978 45 2175 F CGATCTACATGATG 255 8 2247 fR A 622 RPOC EC_21 CAGGAGTCGTTCAACTC RPOCEC_222 290 46 2174 F GATCTACATGAT 256 7 2245 R ACGCCATCAGGCCACGCAT 620 RPOCEC_21 RPOC_EC_222 46_2174_TM TCAGGAGTCGTTCAACT 7_2245_TMOD TACGCCATCAGGCCACGCA 363 OD F CGATCTACATGAT 257 R T 621 RPOC EC 21 78_2196_2 TGATTCCGGTGCCCGTG RPOCEC_222 TTGGCCATCAGACCACGCA 51 F GT 258 5 2246 2 R TAC 618 RPOCEC 21 TGATTCTGGTGCCCGTG RPOCEC_222 TTGGCCATCAGGCCACGCA 50 78 2196F GT 259 5 2246 R TAC 619 RPOCEC_22 18_2241 2_ CTTGCTGGTATGCGTGG RPOCEC_231 CGCACCATGCGTAGAGATG 53 F TCTGATG 260 3 2337 2 R AAGTAC 623 RPOC_EC_22 CTGGCAGGTATGCGTGG RPOC-EC_231 CGCACCGTGGGTTGAGATG 52 18 2241 F TCTGATG 261 3 2337 R AAGTAC 624 RPOC_EC_22 RPOCEC_231 18_2241_TM TCTGGCAGGTATGCGTG 3_2337_TMOD TCGCACCGTGGGTTGAGAT 354 OD F GTCTGATG 262 R GAAGTAC 625 RPOCEC_22 TGGTATGCGTGGTCTGA RPOC_EC_232 TGCTAGACCTTTACGTGCA 958 23 2243 F TGGC 263 9 2352 R CCGTG 626 RPOC EC_23 TGCTCGTAAGGGTCTGG RPOCEC_238 TACTAGACGACGGGTCAGG 960 34 2357 F CGGATAC 264 0 2403 R TAACC 627 RPOCEC_80 CGTCGTGTAATTAACCG RPOCEC_865 ACGTTTTTCGTTTTGAACG 55 8 833 2 F TAACAACCG 265 891 R ATAATGCT 629 RPOCEC_80 CGTCGGGTGATTAACCG RPOCEC_865 GTTTTTCGTTGCGTACGAT 54 8 833 F TAACAACCG 266 889 R GATGTC 628 RPOCEC_91 TATTGGACAACGGTCGT RPOC_EC_100 TTACCGAGCAGGTTCTGAC 961 7 938 F CGCGG 267 9 1034 R GGAAACG 607 RPOC_EC_91 TCTGGATAACGGTCGTC RPOCEC_100 TCCAGCAGGTTCTGACGGA 959 8 938 F GCGG 268 9 1031 R AACG 606 RPOC_EC_99 CAAAGGTAAGCAAGGAC RPOCEC_103 CGAACGGCCAGAGTAGTCA 57 3 1019 2 F GTTTCCGTCA 269 6 1059 2 R ACACG 608 RPOCEC_99 CAAAGGTAAGCAAGGTC RPOC_EC_103 CGAACGGCCTGAGTAGTCA 56 3 1019 F GTTTCCGTCA 270 6 1059 Rt ACACG 609 SP101_SPET AACCTTAATTGGAAAGA SP101_SPET1 CCTACCCAACGTTCACCAA 75 11 1 29 F AACCCAAGAAGT 271 1 92 116 R GGGCAG 676 SPl1_SPET SP101_SPET1 11_1_29_TM TAACCTTAATTGGAAAG 1_92_116_TM TCCTACCCAACGTTCACCA 446 OD F AAACCCAAGAAGT 272 OD R AGGGCAG 677 SPlOlSPET SPlolSPETI 11_1154_11 CAATACCGCAACAGCGG 1_1251_1277 GACCCCAACCTGGCCTTTT 85 79 F TGGCTTGGG 273 R GTCGTTGA 630 SPlolSPET SP101 SPET1 11_115411 TCAATACCGCAACAGCG 1_1251_1277 TGACCCCAACCTGGCCTTT 424 79 TMOD F GTGGCTTGGG 274 TMOD R TGTCGTTGA 631 SP101SPET 11_118_147 GCTGGTGAAAATAACCC SP101_SPET1 TGTGGCCGATTTCACCACC 76 F AGATGTCGTCTTC 275 1 213 238 R TGCTCCT 644 SP101 SPET SP101_SPET1 11_118_147 TGCTGGTGAAAATAACC 1_213_238_T TTGTGGCCGATTTCACCAC 425 TMOD F CAGATGTCGTCTTC 276 MOD R CTGCTCCT 645 86 SP101 SPET CGCAAAAAAATCCAGCT 277 SP101 SPETI AAACTATTTTTTTAGCTAT 632 -35 11 1314_13 ATTAGC 1_1403_1431 ACTCGAACAC 36 F R SP101SPET SP101_SPET1 11_1314_13 TCGCAAAAAAATCCAGC 1_1403_1431 TAAACTATTTTTTTAGCTA 426 36 TMOD F TATTAGC 278 TMOD R TACTCGAACAC 633 SP101 SPET SP101_SPET1 11 1408 14 CGAGTATAGCTAAAAAA 1_1486_1515 GGATAATTGGTCGTAACAA 87 37 F ATAGTTTATGACA 279 R GGGATAGTGAG 634 SP101_SPET SPlOlSPETI 11_1408_14 TCGAGTATAGCTAAAAA 1_1486_1515 TGGATAATTGGTCGTAACA 427 37 TMOD F AATAGTTTATGACA 280 TMOD R AGGGATAGTGAG 635 SP101 SPET SP101_SPETI 11_1688_17 CCTATATTAATCGTTTA 1_1783_1808 ATATGATTATCATTGAACT 88 16 F CAGAAACTGGCT 281 R GCGGCCG 636 SPl1 SPET SP101_SPETI 11 1688 17 TCCTATATTAATCGTTT 1_1783_1808 TATATGATTATCATTGAAC 428 16~TMOD-F ACAGAAACTGGCT 282 TMOD R TGCGGCCG 637 SP1l SPET SP101_SPET1 11 1711 17 CTGGCTAAAACTTTGGC 1_1808_1835 GCGTGACGACCTTCTTGAA 89 33 F AACGGT 283 R TTGTAATCA 638 SP1l SPET SP101_SPET1 11_1711_17 TCTGGCTAAAACTTTGG 1_1808_1835 TGCGTGACGACCTTCTTGA 429 33 TMOD F CAACGGT 284 TMOD R ATTGTAATCA 639 SPlolSPET SP101_SPET1 11 1807_18 ATGATTACAATTCAAGA 11901_1927 TTGGACCTGTAATCAGCTG 90 35 F AGGTCGTCACGC 285 R AATACTGG 640 SPlOlSPET SP101_SPET1 11_1807_18 TATGATTACAATTCAAG 1_1901_1927 TTTGGACCTGTAATCAGCT 430 35 TMOD F AAGGTCGTCACGC 286 TMOD R GAATACTGG 641 SPlOlSPET SP101SPET1 11_1967_19 TAACGGTTATCATGGCC 1 2062 2083 ATTGCCCAGAAATCAAATC 91 91 F CAGATGGG 2B7 R. ATC 642 SP101 SPET SP101 SPETI 11_1967_19 TTAACGGTTATCATGGC 1_2062_2083 TATTGCCCAGAAATCAAAT 431 91 TMOD F CCAGATGGG 288 TMOD R CATC 643 SP101 SPET 11_216_243 AGCAGGTGGTGAAATCG SP101_SPETI TGCCACTTTGACAACTCCT 77 F GCCACATGATT 289 1 308 333 R GTTGCTG 654 SP101 SPET SP101_SPET1 11_216_243 TAGCAGGTGGTGAAATC 1_308_333_T TTGCCACTTTGACAACTCC 432 TMOD F GGCCACATGATT 290 MOD R TGTTGCTG 655 SP101 SPET SP101_SPET1 11 2260_22 CAGAGACCGTTTTATCC 1_2375_2397 TCTGGGTGACCTGGTGTTT 92 83-F TATCAGC 291 R TAGA 646 SP101 SPET SP101_SPET1 112260_22 TCAGAGACCGTTTTATC 1_2375_2397 TTCTGGGTGACCTGGTGTT 433 83 TMOD F CTATCAGC 292 TMOD R TTAGA 647 SP101_SPET SP101_SPET1 11_2375_23 TCTAAAACACCAGGTCA 1_2470_2497 AGCTGCTAGATGAGCTTCT 93 99 F CCCAGAAG 293 R GCCATGGCC 648 SP1l SPET SP101_SPET1 11_2375_23 TTCTAAAACACCAGGTC 1_2470_2497 TAGCTGCTAGATGAGCTTC 434 99 TMOD F ACCCAGAAG 294 TMOD R TGCCATGGCC 649 SP101 SPET SP101_SPET1 11 2468 24 ATGGCCATGGCAGAAGC 1_2543_2570 CCATAAGGTCACCGTCACC 94 87 F TCA 295 R ATTCAAAGC 650 SP101 SPET SP101 SPET1 11_2468_24 TATGGCCATGGCAGAAG 1_2543 2570 TCCATAAGGTCACCGTCAC 435 87 TMOD F CTCA 296 TMOD R CATTCAAAGC 651 SP101 SPET 11_266_295 CTTGTACTTGTGGCTCA SPlOlSPET1 GCTGCTTTGATGGCTGAAT 78 F CACGGCTGTTTGG 297 1 355 380 R CCCCTTC 661 SP101 SPET SP101_SPET1 11_266_295 TCTTGTACTTGTGGCTC 1_355_380_T TGCTGCTTTGATGGCTGAA 436 TMOD F ACACGGCTGTTTGG 298 MOD R TCCCCTTC 662 SPlOlSPET SP1Ol SPET1 11_2961_29 ACCATGACAGAAGGCAT 1_3023_3045 GGAATTTACCAGCGATAGA 95 84 F TTTGACA 299 R CACC 652 SPlOlSPET SP101 SPET1 11_2961_29 TACCATGACAGAAGGCA 1_3023_3045 TGGAATTTACCAGCGATAG 437 84 TMOD F TTTTGACA 300 TMOD R ACACC 653 SP101 SPET SP101_SPET1 11 3075 31 GATGACTTTTTAGCTAA 1_3168 3196 AATCGACGACCATCTTGGA 96 03 F TGGTCAGGCAGC 301 R. AAGATTTCTC 656 438 SP101 SPET TGATGACTTTTTAGCTA 302 SP101 SPET1 TAATCGACGACCATCTTGG 657 -36 11 3075 31 ATGGTCAGGCAGC 1_3168_3196 AAAGATTTCTC 03 TMOD F TMOD R SP1O1SPET sPlOlSPETI 11 3085 31 TAGCTAATGGTCAGGCA 1_3170 3194 TCGACGACCATCTTGGAAA 448 04-F GCC 303 R GATTTC 658 SP10 _SPET 11_322_344 GTCAAAGTGGCACGTTT SP101SPET1 79 F ACTGGC 304 1 423 441 R ATCCCCTGCTTCTGCTGCC 665 SP101_SPET SP101_SPET1 11 322 344 TGTCAAAGTGGCACGTT 1_423_441_T TATCCCCTGCTTCTGCTGC 439 TROD T TACTGGC 305 MOD R C 666 SP101 SPET SP101_SPET1 11_3386_34 AGCGTAAAGGTGAACCT 1_3480_3506 CCAGCAGTTACTGTCCCCT 97 03 F T 306 R CATCTTTG 659 SP101 SPET SP101_SPET1 11 3386_34 TAGCGTAAAGGTGAACC 1_34860 3506 TCCAGCAGTTACTGTCCCC 440 03 TMOD F TT 307 TMOD R TCATCTTTG 660 SP101 SPET SP101_SPET1 11_3511_35 GCTTCAGGAATCAATGA 1_3605_3629 GGGTCTACACCTGCACTTG 98 35 F TGGAGCAG 308 R CATAAC 663 SP101 SPET SP101_SPET1 11_3511_35 TGCTTCAGGAATCAATG 1_3605_3629 TGGGTCTACACCTGCACTT 441 35 TMOD F ATGGAGCAG 309 TMOD R GCATAAC 664 SP101SPET 11_356_387 GGGGATTCAGCCATCAA SP101_SPET1 CCAACCTTTTCCACAACAG 80 F AGCAGCTATTGAC 310 1 448 473 R AATCAGC 668 SP101 SPET SP101_SPET1 11_358_387 TGGGGATTCAGCCATCA 1_448_473_T TCCAACCTTTTCCACAACA 442 TMOD F AAGCAGCTATTGAC 311 MOD R GAATCAGC 669 SP101_SPET 11_364_385 TCAGCCATCAAAGCAGC SP101_SPET1 TACCTTTTCCACAACAGAA 447 F~ ~ TATTG 312 1 448 471 R TCAGC 667 SP101_SPET 11_600_629 CCTTACTTCGAACTATG SP101_SPET1 CCCATTTTTTCACGCATGC 81 F AATCTTTTGGAAG 313 1 686 714 R TGAAAATATC 670 SPl1 SPET SP101_SPET1 11_600 629 TCCTTACTTCGAACTAT 1 686_714_T TCCCATTTTTTCACGCATG 443 TMOD F GAATCTTTTGGAAG 314 MOD R CTGAAAATATC 671 SP101 SPET 11 658 684 GGGGATTGATATCACCG SP101_SPET1 GATTGGCGATAAAGTGATA 82 F ATAAGAAGAA 315 1 756 784 R TTTTCTAAAA 672 SPlOlSPET SP101_SPET1 11_658_684 TGGGGATTGATATCACC 1_756_784_T TGATTGGCGATAAAGTGAT 444 TMOD F GATAAGAAGAA 316 MOD R ATTTTCTAAAA 673 SPlOlSPET 11 776 801 TCGCCAATCAAAACTAA SP101_SPETI GCCCACCAGAAAGACTAGC 83 F GGGAATGGC. 317 1 871 896 R AGGATAA 674 SP101_SPET SP101_SPET1 11_776 801 TTCGCCAATCAAAACTA 1_871_896_T TGCCCACCAGAAAGACTAG 445 TMOD F AGGGAATGGC 318 MOD a CAGGATAA 675 SP101SPET SP101_SPET1 11 893 921 GGGCAACAGCAGCGGAT 1_988_1012 CATGACAGCCAAGACCTCA 84 F TGCGATTGCGCG 319 R CCCACC 678 SP101 SPET SP101_SPET1 11 893 921 TGGGCAACAGCAGCGGA 1_988_1012_ TCATGACAGCCAAGACCTC 423 TMOD F TTGCGATTGCGCG 320 TMOD R ACCCACC 679 SSPE-BA_11 TCAAGCAAACGCACAAT SSPEBA_196 TTGCACGTCTGTTTCAGTT 706 4 137 F CAGAAGC 321 222 R GCAAATTC 683 SSPEBA 11 TCAAGCAAACGCACAAC SSPEBA_196 TTGCACGTU 4 C"GTTTCAGT 612 4 137P F aUAGAAGC 321 2229 R TGCAAATTC 684 SSPE BA 11 CAAGCAAACGCACAATC SSPE_BA_197 TGCACGTCTGTTTCAGTTG 58 5 137 F~ AGAAGC 322 222 R CAAATTC 686 SSPE BA 11 5_137TMOD TCAAGCAAACGCACAAT SSPEBA_197 TTGCACGTCTGTTTCAGTT 355 F CAGAAGC 321 222 TMOD R GCAAATTC 687 SSPE BA 12 SSPE_BA_197 TCTGTTTCAGTTGCAAATT 215 1 137 F AACGCACAATCAGAAGC 323 216 R C 685 SSPE BA 12 TGCACAATCAGAAGCTA SSPEBA_202 TTTCACAGCATGCACGTCT 699 3 153 F AGAAAGCGCAAGCT 324 231 R GTTTCAGTTGC 688 SSPE BA 14 TGCAAGCTTCTGGTGCT SSPEBA_242 TTGTGATTGTTTTGCAGCT 704 6 168 F AGCATT 325 267 R GATTGTG 689 SSPE BA 15 TGCTTCTGGTGCTAGCA SSPEBA_243 TGATTGTTTTGCAGCTGAT 702 0 168 F TT 326 264 R TGT 691 SSPE BA 15 TGCTTCTGGCGUCAG ' SSPE BA_243 TGATTGTTTTGU*AGUaTGA 610 0 1689 F UATT 326 264P R C'C*GT 691 -37 SSPEBA_15 SSPEBA_243 700 6 168 F TGGTGCTAGCATT 327 255 R TGCAGCTGATTGT 690 SSPEBA_15 SSPEBA_243 608 6 168P F TGGCaGU*C"AGU*ATT 327 255P R TGUAGUTGAC*C*GT 690 SSPE BA 63 TGCTAGTTATGGTACAG SSPEBA_163 TCATAACTAGCATTTGTGC 705 89 F AGTTTGCGAC 328 191 R TTTGAATGCT 682 SSPEBA_72 TGGTACAGAGTTTGCGA SSPEBA_163 TCATTTGTGCTTTGAATGC 703 89 F C 329 182 R T 681 SSPE BA 72 TGGTAUAGAGCaC*C"G SSPEBA_163 TCATTTGTGCC*C'C"GAAC 611 89P F UaGAC 329 182P R 'GU*T 681 SSPEBA75 SSPEBA_163 701 89 F TACAGAGTTTGCGAC 330 177 R TGTGCTTTGAATGCT 680 SSPEBA_75 TAUAGAGCaCaCaCGUaG SSPEBA_163 609 89P F AC 330 177P R TGTGCCaC*CaGAACaGU'T 680 TOXR VBC 1 TCGATTAGGCAGCAACG TOXR VBC_22 TTCAAAACCTTGCTCTCGC 1099 35 158 F AAAGCCG 331 1 246 R CAAACAA 692 TRPE AY094 TRPEAY0943 355 1064_1 TCGACCTTTGGCAGGAA 55_1171_119 TACATCGTTTCGCCCAAGA 905 086 F CTAGAC 332 6 R TCAATCA 693 TRPEAY094 TRPE_AY0943 355_1278_1 TCAAATGTACAAGGTGA 55_1392 141 TCCTCTTTTCACAGGCTCT 904 303 F AGTGCGTGA 333 8 R ACTTCATC 694 TRPEAY094 -TRPEAY0943 355 1445 1 TGGATGGCATGGTGAAA 55_1551_158 TATTTGGGTTTCATTCCAC 903 471 F TGGATATGTC 334 0 R TCAGATTCTGG 695 TRPE AY094 TRPEAY0943 355 1467_1 ATGTCGATTGCAATCCG 55_1569_159 TGCGCGAGCTTTTATTTGG 902 491 F TACTTGTG 335 2 R GTTTC 696 TRPEAY094 TRPEAY0943 355 666 68 GTGCATGCGGATACAGA 55_769_791 TTCAAAATGCGGAGGCGTA 906 8 F GCAGAG 336 R TGTG 697 TRPE AY094 TRPEAY0943 355 757 77 TGCAAGCGCGACCACAT 55_864_883 TGCCCAGGTACAACCTGCA 907 6 F ACG 337 R T 698 TUFBEC_22 GCACTATGCACACGTAG TUFBEC_284 TATAGCACCATCCATCTGA 114 5 251 F ATTGTCCTGG 338 309 R GCGGCAC 706 TUFB EC 23 TTGACTGCCCAGGTCAC TUFB EC_283 GCCGTCCATTTGAGCAGCA 60 9 259 2 F GCTG 339 303 2 R- CC 704 TUFBEC 23 TAGACTGCCCAGGACAC TUFB_EC_283 GCCGTCCATCTGAGCAGCA 59 9 259 F GCTG 340 303 R CC 705 TUFB EC 25 TGCACGCCGACTATGTT TUFBEC_337 TATGTGCTCACGAGTTTGC 942 1 278 F AAGAACATGAT 341 360 _R GGCAT 707 TUFB EC 27 TGATCACTGGTGCTGCT TUFBEC_337 TGGATGTGCTCACGAGTCT 941 5 299 F CAGATGGA 342 362 R GTGGCAT 708 TUFBEC_75 AAGACGACCTGCACGGG TUFBEC_849 117 7 774 F C 343 867 R GCGCTCCACGTCTTCACGC 709 TUFB EC 95 CCACACGCCGTTCTTCA TUFBEC_103 GGCATCACCATTTCCTTGT 293 7 979 F ACAACT 344 4 1058 R CCTTCG 700 TUFB EC 95 TUFB_EC_103 7 97_ TMOD TCCACACGCCGTTCTTC 4_1058_TMOD TGGCATCACCATTTCCTTG 367 Pi AACAACT 345 pI TCCTTCG 701 TUFBEC_97 AACTACCGTCCTCAGTT TUFBEC_104 GTTGTCACCAGGCATTACC 62 6 1000 2 F CTACTTCC 346 5 1068 2 R ATTTC 702 TUFB EC 97 AACTACCGTCCGCAGTT TUFBEC_104 GTTGTCGCCAGGCATAACC 61 6 1000 F CTACTTCC 347 5 1068 R ATTTC 703 TUFB EC 98 CCACAGTTCTACTTCCG TUFBEC_103 TCCAGGCATTACCATTTCT 63 5 1012 i TACTACTGACG 348 3 1062 R ACTCCTTCTGG 699 VALSEC 11 CGTGGCGGCGTGGTTAT VALS_EC_119 ACGAACTGGATGTCGCCGT 225 05 1124 F CGA 349 5 1214 R T 710 VALSEC 11 CGTGGCGGCGTGGTTAT VALSEC_119 CGGTACGAACTGGATGTCG 71 05 1124 F CGA 349 5 1218 R. CCGTT 711 VALS EC 11 VALS_EC_119 05_1124TM TCGTGGCGGCGTGGTTA 5_1218_TMOD TCGGTACGAACTGGATGTC 358 OD F TCGA 350 R GCCGTT 712 VALSEC_11 TATGCTGACCGACCAGT VALS EC_123 TTCGCGCATCCAGGAGAAG 965 28 1151 F GGTACGT 351 1 1257 p. TACATGTT 713 VALSEC_18 CGACGCGCTGCGCTTCA VALSEC_192 GCGTTCCACAGCTTGTTGC 112 33 1850 F C 352 0 1943 R AGAAG 714 VALSEC 19 CTTCTGCAACAAGCTGT VALSEC_194 TCGCAGTTCATCAGCACGA 116 20 1943 F GGAACGC 353 8 1970 R AGCG 715 VALSEC_61 ACCGAGCAAGGAGACCA VALS_EC_705 TATAACGCACATCGTCAGG 295 0 649 F GC 354 727 R GTGA 716 WAAA 29692 TCTTGCTCTTTCGTGAG WAAA Z96925 CAAGCGGTTTGCCTCAAAT 931 5 2 9 F TTCAGTAAATG 355 115 138 R AGTCA 717 932 WAAA Z9692 TCGATCTGGTTTCATGC 356 WAAA 796925 TGGCACGAGCCTGACCTGT 718 - 38 5_286 311 TGTTTCAGT 394_412_R SF [0088] Primer pair name codes and reference sequences are shown in Table 2. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name includes coordinates with respect to a reference sequence defined by an extraction of a 5 section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label "no extraction." Where "no extraction" is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the "Gene Name" column. Table 2: Primer Name Codes and Reference Sequences Organism Extraction Primer Reference Extracted gene or entire name Gen~ank coordinates of gi gene code Gene Name gi number number SEQ ID NO: 16S rRNA (16S Escherichia 719 ribosomal RNA coli 16S EC gene) 16127994 4033120..4034661 23S rRNA (23S Escherichia 720 ribosomal RNA coll 23S EC gene) 16127994 4166220..4169123 capC (capsule Bacillus Complement 721 CAPC BA biosynthesis gene) anthracis 6470151 (55628..56074) cya (cyclic AMP Bacillus Complement 722 CYA BA gene) anthracis 4894216 (154288..156626) dnaK (chaperone Escherichia 723 DNAK EC dnaK gene) coll 16127994 12163. .14079 groL (chaperonin Escherichia 724 GROL EC groL) coli 16127994 4368603..4370249 hflb (cell Escherichia 725 division protein coll Complement HFLB EC peptidase ftsH) 16127994 (3322645..3324576) infB (protein Escherichia 726 chain initiation coll Complement INFEB EC factor infB gene) 16127994 (3310983..3313655) lef (lethal Bacillus Complement 727 LEF BA factor) anthracis 21392688 (149357..151786) pag (protective Bacillus 728 PAG BA antigen) anthracis 21392688 143779. .146073 rplB (50S Escherlchia 729 ribosomal protein coll RPLB EC L2) 16127994 3449001..3448180 rpoB (DNA-directed Escherichia 730 RNA polymerase coll Complement RPOB EC beta chain) 6127994 4178823..4182851 rpoC (DNA-directed Escherichia 731 RNA polymerase coll RPOC EC beta' chain) 16127994 4182928..4187151 SP101ET Concatenation SPET_1 comprising: Artificial 732 1 Sequence* - 15674250 gki (glucose partial gene Complement kinase) sequences of (1258294..1258791) Streptococcus gtr (glutamine pyogenes complement transporter (1236751. .1237200) protein) -r! (glutamate 312732. .313169 racemase) mutS (DNA mismatch Complement -39 repair protein) (1787602. .1788007) xpt (xanthine 930977..931425 phosphoribosyl transferase) yqiL (acetyl-CoA- 129471..129903 acetyl transferase) tkt 1391844..1391386 (transketolase) espE (small acid- 733 soluble spore Bacillus SSPE BA protein) anthracis 30253828 226496..226783 tufB (Elongation Escherichia 734 TUFB EC factor Tu) coli 16127994 4173523..4174707 vals (Valyl-tRNA Escherichia Complement 735 VALS EC synthetase) coli 16127994 (4481405..447B550) aSpS (Aspartyl- Escherichia 16127994 complement(19467 77 .. 736 ASPS EC tRNA synthetase) coli 1948546) 2996286 No extraction - CAF1 AF cafl (capsular Yersinia GenBank coordinates 0539747 protein caf1) pests used INV U22 Yersinia 1256565 74..3772 737 457 inv (invasin) pestis ' Y. pests specific 16120353 No extraction - chromosomal genes GenBank coordinates LL NCO - difference Yersinia used 31743 region pestis BONTA X BoNT/A (neurotoxin Clostridium 40381 77..3967 738 52066 type A) botulinum 2791983 No extraction - 739 MECA Y1 mecA methicillin Staphylococcus GenBank- coordinates 4051 resistance gene aureus used trpE (anthranilate 20853695 No extraction TRPE AY synthase (large Acinetobacter GenBank coordinates 094355 component)) baumanil used 740 9965210 No extraction RECA AF recA (recombinase Acinetobacter GenBank coordinates 251469 A) baumanli used 741 4240540 No extraction GYRA AF gyrA (DNA gyrase Acinetobacter GenBank coordinates 100557 subunit A) baumanii used 742 4514436 No extraction GYRBAB gyrB (DNA gyrase Acinetobacter GenBank coordinates 008700 subunit B) baumanil used 743 waaA (3-deoxy-D- 2765828 No extraction WAAA Z9 manno-octulosonic- Acinatobacter GenBank coordinates 6925 acid transferase) baumanil used 744 Concatenation comprising: Artificial Sequence* partial gene tkt sequences of 1569415..1569873 (transketolase) Campylobacter CJSTCJ jejuni glyA (serine 367573..368079 hydroxymethyltrans ferase) 15791399 gitA (citrate complement synthase) (1604529..1604930) aspA (aspartate 96692..97168 ammonia lyase) 745 ginA (glutamine complement synthase) (657609..658085) pgm (phosphoglycerate 327773..328270 mutase) -40 uncA (ATP 112163..112651 synthetase alpha chain) RNASEP_ Base P Bordetella 33591275 Complement BDP (ribonuclease P) pertussIs (3226720..3227933) 746 INASEP EWase P Burkholderia 53723370 Complement BKM (ribonuclease P) mallei (2527296..2528220) 747 BNASEP_ mase P Bacillus 16077068 Complement BS (ribonuclease P) subtilis (2330250..2330962) 748 I1NASEP_ RNase P Clostridium 19308982 Complement CLB (ribonuclease P) perfringens (2291757..2292584) 749 RNASEP MKase P Escherichia 16127994 Complement EC (ribonuclease P) colil (3267457..3268233 750 RNASEP KMase P Rickettsia 15603881 complement(605276..6 RKP (ribonuclease P) prowazekil 06109) 751 RNASEP RNase P Staphylococcus 15922990 complement(1559869.. SA . (ribonuclease P) aureus 1560651) 752 RNASEP_ Mase P Vibrio 15640032 complement(2580367.. VBC (ribonuclease P) cholerae 2581452) 753 icd (isocitrate Coxiella 29732244 complement(1143867.. ICD CXB dehydrogenase) burnetil 1144235) 754 multi-locus Aclnetobacter 29732244 IS1111A insertion baumannii IS1111A element No extraction ompA (outer Rickettsia 40287451 OMPA AY membrane protein prowazekii 485227 A) No extraction 755 ompB (outer Rickettsia 15603881 OMPBRK membrane protein prowazekil complement(881264..8 P B) 86195) 756 GLTARK gitA (citrate Vibrio 15603881 complement(1062547.. P synthase) cholerae 1063857) 757 toxR Francisella 15640032 TOXR VB (transcription tularensis complement(1047143.. C regulator toxR) 1048024) 758 asd (Aspartate Francisella 56707187 semialdehyde tularensls complement(438608..4 ASD FRT dehydrogenase) 39702) 759 GALEFR galE (UDP-glucose Shigella 56707187 T 4-epimerase) flexneri 809039..810058 760 IPAH SG ipaH (invasion Campylobacter 30061571 F plasmid antigen) jejuni 2210775..2211614 761 Coxlella complement(849317..8 hupB (DNA-binding burnetil 15791399 49819) HUPB CJ protein Hu-beta) 762 Concatenation comprising: Artificial 763 Sequence* partial gene sequences of Acinetobacter baumannll trpE (anthranilate synthase component I)) adk (adenylate Sequenced in-house AB MLST kinase) mutY (adenine glycosylase) fumC (fumarate hydratase) efp (elongation factor p) ppa (pyrophosphate phospho hydratase - 41 [0089] * Note: These artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design. The stretches of arbitrary residues "N"s were added for the convenience of separation of the ; partial gene extractions (IOON for SP101._SPET11 (SEQ ID NO: 732); 5ON for CJSTCJ (SEQ ID NO: 745); and 40N for ABMLST (SEQ ID NO: 763)). [0090] Example 2: DNA isolation and Amplification [00911 Genomic materials from culture samples or swabs were prepared using the DNeasy* 96 to Tissue Kit (Qiagen, Valencia, CA). All PCR reactions are assembled in 50 pl reactions in the 96 well microtiter plate format using a Packard MPII liquid handling robotic platform and MJ Dyad* thermocyclers (MJ research, Waltham, MA). The PCR reaction consisted of 4 units of Amplitaq Gold, lx buffer II (Applied Biosystems, Foster City, CA), 1.5 mM MgC 2 , 0.4 M betaine, 800 pM dNTP mix, and 250 nM of each primer. 'S [00921 The following PCR conditions were used to amplify the sequences used for mass spectrometry analysis: 95C for 10 minutes followed by 8 cycles of 95C for 30 seconds, 48C for 30 seconds, and 72C for 30 seconds, with the 48C annealing temperature increased 0.9C after each cycle. The PCR was then continued for 37 additional cycles of 95C for 15 seconds, 56C for to 20 seconds, and 72C for 20 seconds. [0093] Example 3: Solution Capture Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads [0094] For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, : 25 pl of a 2.5 mg/mL suspension of BioClon amine terminated supraparamagnetic beads were added to 25 to 50 i of a PCR reaction containing approximately 10 pM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed 3x with 50mM ammonium ;o bicarbonate/50% MeOH or 100mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with 25mM piperidine, 25mM imidazole, 35% MeOH, plus peptide calibration standards.

-42 [00951 Example 4: Mass Spectrometry and Base Composition Analysis [00961 The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains 5 the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 pl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC) triggered by the FTICR data station. Samples were injected directly into a 10 1 sample loop integrated with a fluidics handling system that supplies the 100 1 /hr flow rate to the ESI source. Ions were formed via electrospray ionization in a modified Analytica (Branford, CT) source S employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter current flow of dry N 2 was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary ao gate electrode, prior to injection into the trapped ion cell where they were mass analyzed. Ionization duty cycles > 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of IM data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s. [00971 The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOFrM. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOFTm ESI source that is 30 equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source. Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 ps.

-43 [00981 The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening o protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute. ; [00991 Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well for the ribosomal DNA-targeted primers and 100 molecules per well for the protein-encoding gene targets. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Serial No. 60/545,425. [01001 Example 5: De Novo Determination of Base Composition of Amplification Products using Molecular Mass Modified Deoxynucleotide Triphosphates s [0101] Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A = 313.058, G = 329.052, C= 289.046, T = 304.046 - See Table 3), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G ++ A (-15.994) combined 3o with C ++ T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A 27

G

3 0

C

21

T

2 1 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A 2 6

G

3 1 C22T 2 0 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a - 44 molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor. [01021 The present invention provides for a means for removing this theoretical 1 Da uncertainty s factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term "nucleobase" as used herein is synonymous with other terms in use in the art including "nucleotide," "deoxynucleotide," "nucleotide residue," "deoxynucleotide residue," "nucleotide triphosphate (NTP)," or deoxynucleotide triphosphate (dNTP). [01031 Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G -+ A combined with C *+ T event (Table 3). Thus, the same the G +-+ A (-15.994) event combined with 5-Iodo-C ++ T (-110.900) event would result in a molecular mass 5 difference of 126.894. If the molecular mass of the base composition A 2 7

G

30 5-Iodo-C 2

T

21 (33422.958) is compared with A 26

G

31 5-Iodo-C 22

T

2 0 , (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A 27

G

30 5 o Iodo-C 21

T

21 . In contrast, the analogous amplification without the mass tag has 18 possible base compositions. Table 3: Molecular Masses of Natural Nucleobases and the Mass-Modified Nucleobase 5 Iodo-C and Molecular Mass Differences Resulting from Transitions Nucleobase Molecular Mass Transition a Molecular Mass A 313.056 A-->T I -9.012 A 313.058 A-->C -24.012 A 313.058 A-->5-Iodo-C 101.888 A 313.058 A-->G 15.994 T 304.046 T-->A 9.012 T 304.046 T-->C -15.000 T 304.046 T-->5-Iodo-C 110.900 T 304.046 T-->G 25.006 C 289.046 C-->A 24.012 C 289.046 C-->T 15.000 -45 C 289.046 C-->G 40.006 5-Iodo-C 414.946 5-Iodo-C-->A -101.888 5-Iodo-C 414.946 5-Iodo-C-->T -110.900 5-Iodo-C 414.946 5-Iado-C-->G -85.894 G 329.052 G-->A -15.994 G 329.052 G-->T -25.006 G 329.052 G-->C -40.006 G 329.052 G-->5-Iodo-C 85.894 101041 Example 6: Data Processing [01051 Mass spectra of bioagent identifying amplicons are analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the g mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer. [01061 The algorithm emphasizes performance predictions culminating in probability-of detection versus probability-of-false-alarm plots for conditions involving complex backgrounds I 0 of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature IS produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. the maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a ao running-sum estimate of the noise-covariance for the cleaned up data. [01071 The amplitudes of all base compositions of bioagent identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this D5 two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product -46 corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction. [01081 Example 7: Use of Broad Range Survey and Division Wide Primer Pairs for 5 Identification of Bacteria in an Epidemic Surveillance Investigation [0109] This investigation employed a set of 16 primer pairs which is herein designated the "surveillance primer set" and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus clade primer pair. The surveillance primer set is shown in Table 4 and consists of primer pairs originally listed in Table 1. This surveillance set comprises primers o with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are is primer pairs 17 and 118. Table 4: Bacterial Primer Pairs of the Surveillance Primer Set Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer No. (SEQ ID (SEQ ID NO:) NO:) 346 16SEC_713_732_TMOD_F 27 16SEC_789_809_THOD_R 389 16S rRNA 10 168_EC_713_732 F 26 16S_EC_789_809 388 16S rRNA 347 16SEC_785_806_TMODF 30 16SEC_880_897_TMODR 392 16S rRNA 11 16S EC 785 806 F 29 16S EC 880 897 R 391 16S rRNA 348 16SEC_960_981_TMODF 38 16S_EC_1054_1073_TMODR 363 16S rRNA 14 16S EC 960 981 F 37 16S EC 1054 1073 R 362 16S rRNA 349 23SEC_1826_1843_THODF 49 23SEC_1906_1924_TMODR 405 23S rRNA 16 23S EC 1826 1843 F 48 23S EC 1906 1924 R 404 23S rRNA 352 INFBEC_1365_1393_TMODF 161 INFB_EC_1439_1467_TMODR 516 infB 34 INFE EC 1365 1393 F 160 INFB EC 1439 1467 R 515 infB 354 RPOCEC_2218_2241_TMODF 262 RPOCEC_2313_2337_TMODR 625 rpoC 52 RPOC EC 2218 2241 F 261 RPOC EC 2313 2337 R 624 rpoC 355 SSPEBA_115_137_TMODF 321 SSPEBA_197_222_TMODR 687 sapE 58 SSPE BA 115 137 F 322 SSPE BA 197 222 R 686 sspE 356 RPLB_EC_650_679_TMOD_F 232 RPLBEC_739_762_TMODR 592 rplB 66 RPLB EC 650 679 F 231 RPLB EC 739 762 R 591 rplB 358 VALSEC_1105_1124_TMODF 350 VALSEC_1195_1218_TMODR 712 valS 71 VALS EC 1105 1124 F 349 VALS EC 1195 1218 R 711 valS 359 RPOBEC 1845 1866_TMODF 241 RPOBEC_1909_1929_TMODR 597 rpoB 72 RPOB EC 1845 1866 F 240 RPOB EC 1909 1929 R 596 rpoB 360 23SEC_2646_2667_TMODF 60 23SEC_2745_2765_TMODR 416 23S rRNA 118 23SEC_2646_2667_F 59 23SEC_2745_2765_R 415 23S rRNA 17 238 EC 2645 2669 F 58 23S EC 2744 2761 R 414 23S rRNA -47 361 16S_EC_1090_1111_2_TMOD_F 5 16SEC_1175_1196_TMODR 370 16S rRNA 3 16S EC 1090 1111 2 F 6 16S EC 1175 1196 R 369 16S rRNA 362 RPOBEC_3799_3821_TMODF 245 RPOB_EC_3862_3888_TMODR 603 rpoB 289 RPOB EC 3799 3821 F 246 RPOB EC 3862 3888 R 602 rpoB 363 RPOCEC_2146_2174_TMOD_F 257 RPOCEC_2227_2245_THODR 621 rpoC 290 RPOC EC 2146 2174 F 256 RPOC EC 2227 2245 R 620 rpoC 367 TUFBEC_957_979_TMODF 345 TUFBEC_1034_1058_TMOD_R 701 tufB 293 TUFB EC 957 979 F 344 TUFB EC 1034 1058 R 700 tufB 449 RPLBEC_690_710_F 237 RPLBEC_737_758_R 589 rplB 357 RPLBEC_688_710_TMODF 236 RPLBEC_736_757_TMODR 588 rplB 67 RPLB EC 688 710 F 235 RPLB EC 736 757 R 587 rp1B [0110] The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished S by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying r o amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or 15 two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level. [0111] Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying .o amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill down analysis primers were designed and are listed in Tables 1 and 5. In Table 5 the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which -48 constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Table 5: Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer No. (SEQ ID (SEQ ID NO:) NO:) 350 CAPCBA_274_303_TMODF 98 CAPC_BA_349_376_TMOD_R 452 capC 24 CAPC BA 274 303 F 97 CAPC BA 349 376 R 451 capC 351 CYABA_1353_1379_TMODF 128 CYA_BA_1448_1467_TMOD_R 483 cyA 30 CYA BA 1353 1379 F 127 CYA BA 1448 1467 R 482 cyA 353 LEFBA_756_781_TMODF 175 LEFBA_843_872_TMODR 531 lef 37 LEF BA 756 781 F 174 LEF BA 843 872 R 530 lef [0112] Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 4 5 and the three Bacillus anthracis drill-down primers of Table 5 is shown in Figure 3 which lists common pathogenic bacteria. Figure 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of Figure 3 can be 0 amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, 1.5 bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a a result of enabling broad triangulation identification. [0113] In Tables 6A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is as indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

-49 Table 6A - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348 Primer 346 Primer 347 Primer 348 organism Strain [A G C T] [A G C T] [A G C T] Klebsiella [29 32 25 13] (23 38 28 26] (26 32 28 30] pneumonliae MGH78578 (29 31 25 131* [23 37 28 261* [26 31 28 30]* CO-92 Biovar [29 30 28 29] Yersinia pestis Orientalis [29 32 25 13] [22 39 28 26] (30 30 27 291* KIM5 P12 (Biovar Yersinia pests Mediaevalis) [29 32 25 13] (22 39 28 26] [29 30 28 29] [29 30 28 29] Yersinia pestis 91001 [29 32 25 13] [22 39 28 26] [30 30 27 291* Haemophilus influenzae KW20 [2B 31 23 17] [24 37 25 27] (29 30 28 29] Pseudomonas [26 36 29 24] aeruginosa PA01 [30 31 23 15] (27 36 29 231* [26 32 29 29] Pseudomonas fluorescens Pf0-1 [30 31 23 15] [26 35 29 25] [28 31 28 29] Pseudomonas putida KT2440 [30 31 23 15] [28 33 27 27] [27 32 29 28] Legionella pneumophila Philadelphia-1 [30 30 24 15] [33 33 23 27] [29 28 28 31] Francisella tularensis schu 4 [32 29 22 16] (28 38 26 26] (25 32 28 31] Bordetella pertussis Tohama ' (30 29 24 16] [23 37 30 24] [30 32 30 261 Burkholderia -27 36 31 24] cepacia J2315 [29 29 27 14] [27 32 26 29] [20 42 35 191* Burkholderia pseudomallei K96243 [29 29 27 14] [27 32 26 29] [27 36 31 24] Neisseria FA 1090, ATCC gonorrhoeae 700825 [29 28 24 18] [27 34 26 281 (24 36 29 27] Neisseria meningitides MC58 (serogroup B) [29 28 26 16] [27 34 27 27] (25 35 30 26] Neisseria meningitides serogroup C, FAM18 (29 28 26 16] (27 34 27 27] [25 35 30 26] Neisseria meningitides Z2491 (serogroup A) (29 28 26 16] [27 34 27 271 [25 35 30 26] Chlamydophila pneumoniae TW-183 (31 27 22 191 NO DATA [32 27 27 29] Chlamydophila pneumoniae AR39 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophila pneumoniae CWLO29 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophila pneumoniae J138 [31 27 22 19] NO DATA (32 27 27 29] Corynebacterium diphtheriae' NCTC13129 (29 34 21 151 (22 38 31 25] [22 33 25 34] Mycobacterium avium k10 [27 36 21 15] (22 37 30 28] (21 36 27 30] Mycobacterium avium 104 [27 36 21 15] (22 37 30 28] [21 36 27 30] Mycobacterium tuberculosis CSD#93 (27 36 21 15] (22 37 30 28] (21 36 27 301 Mycobacterium tuberculosis CDC 1551 (27 36 21 15] [22 37 30 281 (21 36 27 30] Mycobacterium tuberculosis H37Rv (lab strain) [27 36 21 15] (22 37 30 28] (21 36 27 30] Mycoplasma pneumoniae M129 [31 29 19 20] NO DATA NO DATA Staphylococcus [30 29 30 29] aureus MRSA252 [27 30 21 21] [25 35 30 26] [29 31 30 29]* Staphylococcus [30 29 30 29] aureus MSSA476 [27 30 21 21] [25 35 30 26] [30 29 29 30]* Staphylococcus [30 29 30 29] aureus COL (27 30 21 21] [25 35 30 26] (30 29 29 301* Staphylococcus [30 29 30 29] aureus Mu50 [27 30 21 21] (25 35 30 26] [30 29 29 301* Staphylococcus [30 29 30 29] aureus MW2 [27 30 21 211 [25 35 30 26] [30 29 29 301* -50 Staphylococcus [30 29 30 29] aureus N315 [27 30 21 21] [25 35 30 26] [30 29 29 301* Staphylococcus [25 35 30 26] [30 29 30 29] aureus NCTC 8325 (27 30 21 21] [25 35 31 261* [30 29 29 30] Streptococcus [24 36 31 25] agalactlae NEM316 [26 32 23 18] (24 36 30 261* [25 32 29 30] Streptococcus equi NC 002955 [26 32 23 18] (23 37 31 25] (29 30 25 32] streptococcus pyogenes MGAS8232 [26 32 23 18] (24 37 30 25] [25 31 29 31] streptococcus pyogenes MGAS315 (26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes SSI-1 [26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes MGAS10394 [26 32 23 18] [24 37 30 25] (25 31 29 31) Streptococcus pyogenes Manfredo (M5) [26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes SF370 (Ml) (26 32 23 181 [24 37 30 25] (25 31 29 31] Streptococcus pneumoniae 670 [26 32 23 18] [25 35 28 28] [25 32 29 30] Streptococcus pneumoniae R6 (26 32 23 181 [25 35 28 28] (25 32 29 30] Streptococcus pneumoniae TIGR4 (26 32 23 18] (25 35 28 28] [25 32 30 29] Streptococcus gordonii NCTC7868 [25 33 23 18] [24 36 31 25] [25 31 29 31] Streptococcus [25 32 29 30] Mitis NCTC 12261 [26 32 23 181 [25 35 30 26] [24 31 35 29]* Streptococcus mutans UA159 (24 32 24 19] [25 37 30 24] (28 31 26 31] Table 6B - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 349,360, and 356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 [25 31 25 221 (33 37 25 27] NO DATA CO-92 Biovar (25 31 27 20] Yersinia pestis Orientalis [25 32 26 20]* [34 35 25 28] NO DATA KIM5 P12 (Biovar (25 31 27 20] Yersinia pestis Mediaevalis) (25 32 26 20]* [34 35 25 28] NO DATA Yersinia pests 91001 (25 31 27 20] [34 35 25 28] NO DATA Haemophilus influenzae KW20 [28 28 25 20) [32 38 25 27] NO DATA Pseudomonas [31 36 27 27] aeruginosa PA01 (24 31 26 20] [31 36 27 281* NO DATA Pseudomonas [30 37 27 28] fluorescens PfM-1 NO DATA (30 37 27 28] NO DATA Pseudomonas putida KT2440 (24 31 26 20] [30 37 27 28] NO DATA Legionella pneumophila Philadelphia-1 [23 30 25 23] [30 39 29 24] NO DATA Francisella tularensis schu 4 [26 31 25 19] [32 36 27 271 NO DATA Bordetella pertussis Tohama I (21 29 24 18] [33 36 26 27] NO DATA Burkholderia cepacia J2315 (23 27 22 20] (31 37 28 26] NO DATA Burkholderia pseudomallel K96243 [23 27 22 20] [31 37 28 26] NO DATA Neisseria gonorrhoeae FA 1090, ATCC 700825 (24 27 24 17] [34 37 25 26] NO DATA Neisseria meningitidis MC58 (serogroup B) [25 27 22 18] [34 37 25 261 NO DATA ?elsseria meningitidis serogroup C, FAM18 [25 26 23 18] [34 37 25 26] NO DATA Melsseria Z2491 (serogroup A) (25 26 23 18] [34 37 25 26] NO DATA - 51 meningitidis Chlamydophila pneumoniae TW-183 (30 28 27 18] NO DATA NO DATA chlamydophila pneumoniae AR39 [30 28 27 18] NO DATA NO DATA Chlamydophila pneumoniae CWL029 (30 28 27 18] NO DATA NO DATA Chlamydophila pneumoniae J138 [30 28 27 18] NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA (29 40 28 25] NO DATA Mycobacterium avium k10 NO DATA [33 35 32 22] NO DATA Mycobacterium avium 104 NO DATA [33 35 32 22] NO DATA mycobacterium tuberculosis CSU#93 NO DATA [30 36 34 22] NO DATA 3Mycobacterum tuberculosis CDC 1551 NO DATA [30 36 34 22] NO DATA mycobacterium tuberculosis H37Rv (lab strain) NO DATA (30 36 34 22] NO DATA Mycoplasma pneumoniae M129 [28 30 24 19) [34 31 29 28] NO DATA Staphylococcus aureus MRSA252 [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus MSSA476 [26 30 25 20] (31 38 24 29] [33 30 31 27] Staphylococcus aureus COL (26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus Mu50 [26 30 25 20] [31 38 24 29] (33 30 31 27] Staphylococcus aureus MW2 [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus N315 [26 30 25 20] [31 38 24 29] (33 30 31 27] Staphylococcus aureus NCTC 8325 [26 30 25 20] (31 38 24 29] [33 30 31 27] Streptococcus agalactiae NEM316 [28 31 22 20] [33 37 24 28] [37 30 28 26] Streptococcus equI NC 002955 [28 31 23 19] [33 38 24 27] (37 31 28 25] Streptococcus pyogenes MGAS8232 [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus pyogenes MGAS315 (28 31 23 19] [33 37 24 28] (38 31 29 23] Streptococcus pyogenes SSI-1 (28 31 23 19] 133 37 24 28] [38 31 29 23] Streptococcus pyogenes MGAS10394 [28 31 23 19] [33 37 24 281 [38 31 29 23] Streptococcus pyogenes Manfredo (M5) [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus [28 31 23 19] pyogenes SF370 (Ml) [28 31 22 201* [33 37 24 28] [38 31 29 23] Streptococcus pneumoniae 670 [28 31 22 20] [34 36 24 28] (37 30 29 25] Streptococcus pneumonlae R6 [28 31 22 20] (34 36 24 29] [37 30 29 25] Streptococcus pneumoniae TIGR4 (28 31 22 20] (34 36 24 28] [37 30 29 25] Streptococcus gordonii NCTC7868 [28 32 23 20] (34 36 24 28] (36 31 29 25] Streptococcus [28 31 22 20] mitis NCTC 12261 (29 30 22 201* [34 36 24 28] [37 30 29 25] Streptococcus mutans UA159 (26 32 23 22] [34 37 24 271 NO DATA -52 Table 6C - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352 Primer 449 Primer 354 Primer 352 organism Strain (A G C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 NO DATA (27 33 36 26] NO DATA CO-92 Biovar yersinia pestis Orientalis NO DATA (29 31 33 29] (32 26 20 25] KIM5 P12 (Biovar Yersinia pests Mediaevalis) NO DATA [29 31 33 29] [32 26 20 25] yersinia pests 91001 NO DATA [29 31 33 291 NO DATA Haemophilus influenzae KW20 NO DATA (30 29 31 32] NO DATA Pseudomonas aeruginosa PA01 NO DATA [26 33 39 24] NO DATA Pseudomonas fluorescens PfM-1 NO DATA [26 33 34 29] NO DATA Pseudomonas putida KT2440 NO DATA (25 34 36 27] NO DATA Legionella pneumophila Philadelphia-1 NO DATA NO DATA NO DATA Francisella tularensis schu 4 NO DATA (33 32 25 32] NO DATA Bordetella pertussis Tohama I NO DATA [26 33 39 24] NO DATA Burkholderia cepacia J2315 NO DATA [25 37 33 27] NO DATA Burkholderia pseudomallei K96243 NO DATA (25 37 34 26] NO DATA Neisseria gonorrhoeae FA 1090, ATCC 700825 (17 23 22 10] (29 31 32 30] NO DATA Neisseria meningitidis MCSB (serogroup B) NO DATA (29 30 32 31] NO DATA Neisseria meningitidis serogroup C, FAM18 NO DATA [29 30 32 31] NO DATA Neisseria meningitidis Z2491 (serogroup A) NO DATA (29 30 32 31] NO DATA Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium k1O NO DATA NO DATA NO DATA Mycobacterium avium 104 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA Mycobacterium tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA Staphylococcus aureus MRSA252 (17 20 21 17] (30 27 30 35] (36 24 19 26] Staphylococcus aureus MSSA476 [17 20 21 17] (30 27 30 35] (36 24 19 26] Staphylococcus aureus COL [17 20 21 17] [30 27 30 35] (35 24 19 271 Staphylococcus aureus Mu50 (17 20 21 17] (30 27 30 35] [36 24 19 26] Staphylococcus aureus MW2 (17 20 21 17] (30 27 30 35] 36 24 19 26] -53 Staphylococcus aureus N315 [17 20 21 17] [30 27 30 35] [36 24 19 26] staphylococcus aureus NCTC 8325 (17 20 21 17] [30 27 30 35] [35 24 19 271 Streptococcus agalactlae NEM316 [22 20 19 14] [26 31 27 38] [29 26 22 28] Streptococcus equi NC 002955 [22 21 19 13] NO DATA NO DATA Streptococcus pyogenes MGAS8232 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes MGAS315 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes SSI-1 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes MGAS10394 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes Manfredo (M5) [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes SF370 (M1) [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pneumoniae 670 [22 20 19 14] [25 33 29 351 [30 29 21 25] Streptococcus pneumoniae R6 (22 20 19 14] [25 33 29 35] [30 29 21 25] Streptococcus pneumoniae TIGR4 [22 20 19 14] [25 33 29 35] [30 29 21 25] Streptococcus gordonii NCTC7868 [21 21 19 14] NO DATA (29 26 22 28] Streptococcus mitis NCTC 12261 (22 20 19 14] [26 30 32 34] NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA Table 6D - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 NO DATA [24 39 33 20] [25 21 24 17] CO-92 Biovar Yersinia pestis Orientalis NO DATA [26 34 35 21] [23 23 19 22] KIMS P12 (Biovar Yersinia pestis Mediaevalis) NO DATA [26 34 35 21) [23 23 19 22] Yersinia pests 91001 NO DATA [26 34 35 21] (23 23 19 22] Haemophilus influenzae KW20 NO DATA NO DATA NO DATA Pseudomonas aeruginosa PA01 NO DATA NO DATA NO DATA Pseudomonas fluorescens Pf0-1 NO DATA NO DATA NO DATA Pseudomonas putida KT2440 NO DATA [21 37 37 21] NO DATA Leglonelle pneumophila Philadelphia-I NO DATA NO DATA NO DATA Franclsella tularensis schu 4 NO DATA NO DATA NO DATA Bordetella pertussis Tohama I NO DATA NO DATA NO DATA Burkholderla cepacia J2315 NO DATA NO DATA NO DATA Burkholderia pseudomallei K96243 NO DATA NO DATA NO DATA Neisseria gonorrhoeae FA 1090, ATCC 700825 NO DATA NO DATA NO DATA Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA Neisseria meningitidis serogroup C, FAM18 NO DATA NO DATA NO DATA - 54 Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA Chlamydophila pneumonlae TW-183 NO DATA NO DATA NO DATA Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium k1O NO DATA NO DATA NO DATA Mycobacterium avium 104 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA Mycobacterium tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA Staphylococcus aureus COL NO DATA NO DATA NO DATA Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA Staphylococcus aureus MW2 NO DATA NO DATA NO DATA Staphylococcus aureus N315 NO DATA NO DATA NO DATA Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA Streptococcus agalactiae NEM316 ' NO DATA NO DATA NO DATA Streptococcus equi NC 002955 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA Streptococcus pyogenes . MGAS315 NO DATA NO DATA NO DATA Streptococcus pyogenes SSI-1 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA Streptococcus pyogenes Manfredo (M5) NO DATA NO DATA NO DATA Streptococcus pyogenes SF370 (Ml) NO DATA NO DATA NO DATA Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA Streptococcus pneumoniae R6 NO DATA NO DATA NO DATA Streptococcus pneumoniae TIGR4 NO DATA NO DATA NO DATA Streptococcus gordonii NCTC7868 NO DATA NO DATA NO DATA Streptococcus mitis NCTC 12261 NO DATA NO DATA NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA -55 Table 6E - Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 362,363, and 367 Primer 362 Primer 363 Primer 367 organism Strain [AGCTA G C T [A GCT Klebsiella pneumoniae MGH78578 [21 33 22 16] [16 34 26 26] NO DATA CO-92 Biovar yersinia pestis Orientalis [20 34 18 20] NO DATA NO DATA KIM5 P12 (Biovar Yersinia pestis Mediaevalis) [20 34 18 20] NO DATA NO DATA. yersinia pestis 91001 [20 34 18 20] NO DATA NO DATA Haemophilus influenzae KW20 NO DATA NO DATA NO DATA Pseudomonas aeruginosa PA01 [19 35 21 17] [16 36 28 22] NO DATA Pseudomonas fluorescens Pf0-1 NO DATA -[18 35 26 23] NO DATA Pseudomonas putida KT2440 NO DATA [16 35 28 23] NO DATA Legionella pneumophila Philadelphia-i NO DATA NO DATA NO DATA Francisella tularensis schu 4 NO DATA NO DATA NO DATA Bordetella pertussis Tohama I [20 31 24 171 [15 34 32 21] [26 25 34 19] Burkholderia cepacia J2315 [20 33 21 18] [15 36 26 25] [25 27 32 20] Burkholderia pseudomallel K96243 [19 34 19 20] [15 37 28 22] [25 27 32 20] Neisseria , gonorrhoeae FA 1090, ATCC 700625 NO DATA NO DATA NO DATA Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA Neisseria meningitidis serogroup C, FAM18 NO DATA NO DATA NO DATA Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium k10 [19 34 23 16] NO DATA (24 26 35 19] Mycobacterium avium 104 [19 34 23 16] NO DATA [24 26 35 19] Mycobacterium tuberculosis CSU#93 [19 31 25 171 NO DATA [25 25 34 20] Mycobacterilum tuberculosis CDC 1551 [19 31 24 18] NO DATA [25 25 34 20] Mycobacterium tuberculosis H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 20] Mycoplasma neumoniae M129 NO DATA NO DATA NO DATA Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA Staphylococcus aureus COL NO DATA NO DATA NO DATA Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA Staphylococcus aureus MW2 NO DATA NO DATA NO DATA Staphylococcus N315 NO DATA NO DATA NO DATA -56 aureus Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA Streptococcus agalactiae NEM316 NO DATA NO DATA NO DATA Streptococcus eqUi NC 002955 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS315 NO DATA NO DATA NO DATA Streptococcus pyogenes SSI-I NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA Streptococcus pyogenes Manfredo (M5) ' NO DATA NO DATA NO DATA Streptococcus pyogenes SF370 (M1) NO DATA NO DATA NO DATA Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA Streptococcus pneumoniae R6 [20 30 19 23] NO DATA NO DATA Streptococcus pneumoniae TIGR4 (20 30 19 23] NO DATA NO DATA Streptococcus gordonil NCTC7868 NO DATA NO DATA NO DATA Streptococcus mitis NCTC 12261 NO DATA NO DATA NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA [0114] Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from November 1 to December 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States 5 since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus -associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and to other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak. [0115] Pure colonies isolated from group A Streptococcus-selective media from all four Is collection periods were analyzed with the surveillance primer set All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in Figure 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair - 57 number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms. 5 [0116] In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 49 and 405) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to o analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 TI 8), Neisseria meningitidis (A25 G27 C22 T1 8), and Haemophilus influenzae (A28 G28 C25 T20) (see Figure 5 and Table 6B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned U.S. Patent Application Serial No: 60/545,425 which S is incorporated herein by reference in its entirety. [01171 Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 232:592) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haemophilus influenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis ) and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes (Figures 3 and 6, Table 6B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample. [01181 The 15 throat swabs from military recruits were found to contain a relatively small set of so microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial - 58 flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci (S. parasangunis, S. vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the s military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease. [01191 Example 8: Drill-down Analysis for Determination of emm-Type of Streptococcus o pyogenes in Epidemic Surveillance [01201 As a continuation of the epidemic surveillance investigation of Example 7, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping 15 genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the ao challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined. [0121] An alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murl), as DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes. 30 These six primer pairs are displayed in Table 7. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

-59 Table 7: Group A Streptococcus Drill-Down Primer Pairs Primer Forward Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair No. (SEQ ID NO:) (SEQ ID NO:) SP101_SPET11_358_387 SP101_SPET11 448_ 442 TMOD_F 311 473_TMOD-R 669 gki 80 SP101_SPET11_358_387 310 SP101_SPET11 448 668 gki F 473 TMOD R SP101_.SPET11_600_629_ SP101_SPET11_686_ 443 TMODF 314 714_TMODR 671 gtr 81 SP101_SPET11_600_629 313 SP101 SPET11_686_ 670 gtr F 714 I SP101_SPETI_1314_133 SP101 SPETI1_1403 426 6_TMODF 278 _1431_TMODR 633 murl 86 SP101_SPET11_1314_133 277 SP101_SPET11_1403 632 murI 6 F 1431 R SP101_SPET11_1807_183 SP101_SPET11_1901 430 5_TMODF 286 _1927_TMODR 641 mutS 90 SP101_SPET11_1807_183 285 SP101_SPET11_1901 640 mutS 5 F 1927 R SP101_SPET11_3075_310 SP101_SPET11_3168 438 3_TMODF 302 _3196_TMODR 657 xpt 96 SP101SPET11_3075_310 301 SP101_SPET11_3168 656 xpt 3 F 3196 R SP101_SPET11_3511_353 SP101_SPET11_3605 441 5_TMODF 309 _3629_TMOD_R 664 yqiL 98 SP101_SPET11_3511_353 308 SP101_SPET11_3605 663 yqiL 5 F 3629 R [01221 The primers of Table 7 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated. 5 [01231 Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 8A-C rows 1-3), all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic 10 strains. Archived samples (Tables 8A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

-60 Table 8A: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430 # of em-type by emn-Gene Location murI muts I n n Mass Year (Primer Pair (Primer Pair instance Spectrometry Sequencing (sample) No. 426) No. 430) 48 3 3 MCRD San A39 G25 C20 T34 A38 G27 C23 T33 2 6 6 Diego 2002 A40 G24 C20 T34 A38 G27 C23 T33 1 28 28 A39 G25 C20 T34 A38 G27 C23 T33 (Cultured) 15 3 ND A39 G25 C20 T34 A38 G27 C23 T33 6 3 3 A39 G25 C20 T34 A38 G27 C23 T33 3 5,58 5 A40 G24 C20 T34 A38 G27 C23 T33 6 6 6 NHRC San A40 G24 C20 T34 A38 G27 C23 T33 1 11 11 Diego- A39 G25 C20 T34 A38 G27 C23 T33 3 12 12 Archive 2003 A40 G24 C20 T34 A38 G26 C24 T33 1 22 22 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 3 25,75 75 A39 G25 C20 T34 A38 G27 C23 T33 4 44/61,82,9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 53,91 91 A39 G25 C20 T34 A38 G27 C23 T33 1 2 2 A39 G25 C20 T34 A38 G27 C24 T32 2 3 3 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 A39 G25 C20 T34 A38 G27 C23 T33 1 6 6 Ft. A40 G24 C20 T34 A38 G27 C23 T33 11 25 or 75 75 Lonard 2003 A39 G25 C20 T34 A38 G27 C23 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 44/61 or 82 1 or 9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 5 or 58 5 A40 G24 C20 T34 A38 G27 C23 T33 3 1 1 A40 G24 C20 T34 A38 G27 C23 T33 2 t S 2003 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 1 28 28 A39 G25 C20 T34 A38 G27 C23 T33 1 3 3 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 A39 G25 C20 T34 A38 G27 C23 T33 3 6 6 A40 G24 C20 T34 A38 G27 C23 T33 1 11 11 Ft. A39 G25 C20 T34 A38 G27 C23 T33 1 13 94** Benning 2003 A40 G24 C20 T34 A38 G27 C23 T33 44/61 or 82 (Cultured) 1 or 9 82 A40 G24 C20 T34 A38 G26 C24 T33 1 5 or 58 58 A40 G24 C20 T34 A38 G27 C23 T33 1 78 or 89 89 A39 G25 C20 T34 A36 G27 C23 T33 2----- Lackland A40 G24 C20 T34 A38 G27 C23 T33 1 - AFB A39 G25 C20 T34 A38 G27 C24 T32 1 81 or 90 ND 2003 A40 G24 C20 T34 A38 G27 C23 T33 1 78 (Throat A38 G26 C20 T34 A38 G27 C23 T33 Swabs) 3*** No detection No detection No detection 7 3 ND A39 G25 C20 T34 A38 G27 C23 T33 1 3 ND MCRD San No detection A38 G27 C23 T33 1 3 ND Diego 2002 No detection No detection 1 3 ND (Throat No detection No detection 2 3 ND Swabs) No detection A38 G27 C23 T33 3 No detection ND No detection No detection - 61 Table 8B: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 # of -m-type by amGene Location xtYL inofance -tyas p by qu (a n Year (Primer Pair (Primer Pair Instances petrometry Sequencing (sample) No. 438) No. 441) 48 3 3 MCRD San A30 G36 C20 T36 A40 G29 C19 T31 2 .6 6 Diego 2002 A30 G36 C20 T36 A40 G29 C19 T31 1 28 28 A30 G36 C20 T36 A41 G28 g18 T32 (Cultured) 15 3 ND A30 G36 C20 T36 A40 G29 C19 T31 6 3 3 A30 G36 C20 T36 A40 G29 C19 T31 3 5,58 5 A30 G36 C20 T36 A40 G29 C19 T31 6 6 6 NHRC San A30 G36 C20 T36 A40 G29 C19 T31 1 11 11 Diego- A30 G36 C20 T36 A40 G29 C19 T31 3 12 12 Archive 2003 A30 G36 C19 T37 A40 G29 C19 T31 1 22 22 (Cultured) A30 G36 C20 T36 A40 G29 C19 T31 3 25,75 75 A30 G36 C20 T36 A40 G29 C19 T31 4 44/61,82,9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 53,91 91 A30 G36 C19 T37 A40 G29 C19 T31 1 2 2 A30 G36 C20 T36 A40 G29 C19 T31 2 3 3 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 A30 G36 C19 T37 A41 G28 C19 T31 1 6 6 Ft. A30 G36 C20 T36 A40 G29 C19 T31 11 25-or-75-- Leonard :125.r75---.Wood 2003 A30 G36 C20 T36 A40 G29 C19 T31 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C19 T37 A40 G29 C19 T31 44/61 or 82 1 or 9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 5 - A30 G36 C20 T36 A40 G29 C19 T31 3 1 1 A30 G36 C19 T37 A40 G29 C19 T31 2 3 3 Ft. Sill 2003 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 (Cultured) A30 G36 C19 T37 A41 G28 C19 T31 1 28 28 A30 G36 C20 T36 A41 G28 C18 T32 1 3 3 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 A30 G36 C19 T37 A41 G28 C19 T31 3 6 6 A30 G36 C20 T36 A40 G29 C19 T31 1 11 11 Ft. A30 G36 C20 T36 A40 G29 C19 T31 1 13 -94** Benning 2003 A30 G36 C20 T36 A41 G28 C19 T31 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C20 T36 A41 G28 C19 T31 1 5 or 58 58 A30 G36 C20 T36 A40 G29 C19 T31 1 78 or 89 89 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 Lkld A30 G36 C20 T36 A40 G29 C19 T31 1 2 AFc A30 G36 C20 T36 A40 G29 C19 T31 1 81 or 90 ND 2003 A30 G36 C20 T36 A40 G29 C19 T31 1 78 (Throat A30 G36 C20 T36 A41 G28 C19 T31 Swabs) 3*** No detection No detection No detection 7 3 ND A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND MCRD San A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND Diego 2002 A30 G36 C20 T36 No detection 1 3 ND (Throat No detection A40 G29 C19 T31 2 3 ND Swabs) A30 G36 C20 T36 A40 G29 C19 T31 3 No detection ND No detection No detection -62 Table 8C: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 # of em-yeb earn-Gene Location kgt masse ene (oation Year (Primer Pair ((Primer Pair Instances pectrome Sequencing (sample) No. 442) No. 443) 48 3 3 MCRD San A32 G35 C17 T32 A39 G28 C16 T32 2 6 6 Diego A31 G35 C17 T33 A39 G28 C15 T33 2002 1 28 28 uA30 G36 C17 T33 A39 G28 C16 T32 15 3 ND (Cultured) A32 G35 C17 T32 A39 G28 C16 T32 6 3 3 A32 G35 C17 T32 A39 G28 C16 T32 3 5,58 5 A30 G36 C20 T30 A39 G28 C15 T33 6NHRC San A31 G35 C17 T33 A39 G28 C15 T33 1 11 11 Diego- A30 G36 C20 T30 A39 G28 C16 T32 3 12 12 Archive 2003 A31 G35 C17 T33 A39 G28 C15 T33 1 22 22 (Cultured) A31 G35 C17 T33 A38 G29 C15 T33 3 25,75 75 A30 G36 C17 T33 A39 G28 C15 T33 4 44/61,82,9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 53,91 91 A32 G35 C17 T32 A39 G28 C16 T32 1 2 2 A30 G36 C17 T33 A39 G28 C15 T33 2 3 3 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A39 G28 C15 T33 1 6 6 Ft. A31 G35 C17 T33 A39 G28 C15 T33 11 25 or 75 75 onard 2003 A30 G36 C17 T33 A39 G28 C15 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C17 T33 A39 G28 C15 T33 44/61 or 82 1 or 9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 5 A30 G36 C20 T30 A39 G28 C15 T33 3 1 1 A30 G36 C18 T32 A39 G28 C15 T33 2 3 3 Ft. Sill 2003 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 (Cultured) A31 G35 C17 T33 A39 G28 C15 T33 1 28 28 A30 G36 C17 T33 A39 G28 C16 T32 1 3 3 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A39 G28 C15 T33 3 6 6 A31 G35 C17 T33 A39 G28 C15 T33 1 11 11 Ft. A30 G36 C20 T30 A39 G28 C16 T32 1 13 94** Benning 2003 A30 G36 C19 T31 A39 G28 C15 T33 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C18 T32 A39 G28 C15 T33 1 5 or 58 58 A30 G36 C20 T30 A39 G28 C15 T33 1 78 or 89 89 A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 Ak d A30 G36 C20 T30 A39 G28 C15 T33 1 2 aFB A30 G36 C17 T33 A39 G28 C15 T33 1 81 or 90 ND 2003 A30 G36 C17 T33 A39 G28 C15 T33 1 78 (Throat A30 G36 C18 T32 A39 G28 C15 T33 Swabs) 3*** No detection No detection No detection 7 3 ND A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND MCRD San No detection No detection 1 3 ND Diego 2002 A32 G35 C17 T32 A39 G28 C16 T32 1 3 ND (Throat A32 G35 C17 T32 No detection 2 3 ND Swabs) A32 G35 C17 T32 No detection 3 No detection ND No detection No detection -63 [0124] Example 9: Design of Calibrant Polynucleotides based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons) [01251 This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 4) and the Bacillus anthracis drill-down set (Table 5). [0126] Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Table 4 (primer names have the designation "TMOD"). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 9. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 722. In Table 9, the forward (_F) or reverse (_R) primer 5 name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16SEC_713_732_TMODF indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic !0 sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 10. The designation "TMOD" in the primer names indicates that the 5' end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5' end of the amplification product, an occurrence whigh may result in miscalculation of base composition as from molecular mass data (vide supra). [0127] The '19 calibration sequences described in Tables 9 and 10 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 741 - which is herein designated a "combination calibration polynucleotide") which was then cloned into a pCR-Blunt vector 30 (Invitrogen, Carlsbad, CA). This combination calibration polynucleotide can be used in conjunction with the primers of Table 9 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in ap amplification -64 reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 783) are indicated in Table 10. Table 9: Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying Amplicons and Corresponding Representative Calibration Sequences Primer Forward Primer Name Forward Revere Primer Name Reverse Calibration Calibration Pair No. Primer Primer Sequence Model Sequence (SEQ ID (SEQ ID Species (SEQ ID NO:) NO:) NO:) 361 16SEC_1090_1111_2_T 5 16SEC_1175_1196_TMODR 370 Bacillus 764 MOD F anthracis 346 16SEC_713_732_TMOD_ 27 16SEC_789_809_TMODR 389 Bacillus 765 F _anthracis 347 16S_EC_785_806_TMOD_ 30 16SEC_880_897_TMODR 392 Bacillus 766 F _anthracls 348 16S_EC_960_981_THOD_ 38 16SEC_1054_1073_THODR 363 Bacillus 767 F anthracis 349 23SEC_1826 1843_TMO 49 23SEC_1906_1924_TMODR 405 Bacillus 768 DF anthracis 360 23S_EC_2646_2667_TMO 60 23SEC_2745_2765_TMODR 416 Bacillus 769 D F anthracis 350 CAPCBA_274_303_TMOD 98 CAPCBA_349_376_TMODR 452 Bacillus 770 F anthracis 351 CYABA_1353_1379_TMO 128 CYABA_1448_1467_TMOD_R 483 Bacillus 771 D F anthracis 352 INFB_EC_1365_1393_TM 161 INFBEC_1439_1467_TMOD_ 516 Bacillus 772 1 OD F R anthracis 353 LEF BA 756 781 TM0D_ 175 LEFBA_843_872_TMODR 531 Bacillus 773 F anthracis 356 RPLBEC_650_679TMOD 232 RPLBEC_739_762_TMODR 592 Clostridium 774 1 F ~_~_~_~_botulinum 449 RPLBEC_690_710_F 237 RPLBEC737_758_R 589 Clostridum 775 botulinum 359 RPOBEC_1845_1866 TM 241 RPOBEC_1909_1929_TMOD_ 597 Yersinia 776 OD F R Pea ti 362 RPOBEC 3799_3821 TM 245 RPOBEC_3862 3888_TMOD_ 603 Burkholderla 777 OD F ~ ~ ~mallei 363 RPOCEC_2146 2174_TM 257 RPOCEC_2227 2245_T40D 621 Burkholderia 778 OD F ~_~_~ R malleI 354 RPOC EC 2218 2241 TM 262 RPOCEC_2313_2337_TMOD 625 Bacillus 779 OD F ~R~_~ anthracla 355 SSPE BA 115 137 TMOD 321 SSPEBA_197_222_THODR 687 Bacillus 780 F ~ anthracis 367 TUFB EC 957_979TMOD 345 TUFB_EC_1034_1058_TMOD_ 701 Burkholderia 781 F R mallel 358 VALSEC_1105_1124_TM 350 VALS_EC_1195_1218_TMOD_ 712 erainia 782 OD F R Fascia Table 10: Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Bacterial Gene Gene Extraction Coordinates Reference GenBank GI No. of Primer Pair Coordinates of Calibration and Species of Genomic or Plasmid Sequence Genomic (G) or Plasmid (P) No. Sequence In Combination Sequence Calibration Polynucleotide (SEQ ID NO: 783) 16S E. coll 4033120..4034661 16127994 (G) 346 16..109 16S E. coll 4033120..4034661 16127994 (G) 347 83..190 16S E. coll 4033120..4034661 16127994 (G) 348 246..353 16S E. coll 4033120..4034661 16127994 (G) 361 368..469 23S H. coll 4166220..4169123 16127994 (G) 349 743..837 23S E. coll 4166220..4169123 16127994 (G) 360 865..981 rpoB Z. 4178823..4182851 16127994 (G) 359 1591..1672 coll. (complement strand) rpoB E. coll 4178823..4182851 16127994 (G) 362 20E1..2167 (complement strand) rpoC R. coll 4182928..4187151 16127994 (G) 354 1810..1926 rpoC E. coll 4182928..4187151 16127994 (G) 363 2183..2279 infB S. coll 3313655..3310983 16127994 (G) 352 1692..1791 (complement strand) tufB E. coll 4173523..4174707 16127994 (G) 367 2400..2498 rplB Z. oCol 3449001..3448180 16127994 (G) 356 1945..2060 rplB E. coll 3449001..3448180 16127994 (G) 449 1986. .2055 valS S. coll 4481405..4478550 16127994 (G) 358 1462..1572 I (complement strand) I -65 capC 56074..55628 (complement 6470151 (P) 350 2517..2616 B. anthracis strand) c y 156626..154288 4894216 (P) 351 1338. .1449 B, anthracis (complement strand) lef 127442..129921 4894216 (P) 353 1121..1234 B. ant)hracis _____________ ___________ a nTspE 226496..226783 30253828 (G) 355 1007-1104 B. anthracis [0128] Example 10: Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes [01291 The process described in this example is shown in Figure 7. The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer s pair number 350 (see Tables 9 and 10) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 3 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the o combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in Figure 8). The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) s and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis. Methods of calculating quantities of molecules based on internal calibration procedures are well known to ao those of ordinary skill in the art. [01301 Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid. [01311 Example 11: Drill-down Genotyping of Campylobacter Species [01321 A series of drill-down primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacterjejuni. The primers are listed in Table 11 with the designation "CJST_CJ." Housekeeping genes to which the primers hybridize 30 and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine -66 hydroxymethyltransferase), gltA (citrate synthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain). Table 11: Campylobacter Drill-down Primer Pairs Primer Fonvard Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair (SEQ ID NO:) (SEQ ID NO:) No. 1053 CJST CJ 1080 1110 F 102 CJST CJ 1166 1198 R 456 gltA 1064 CJST CJ 16B0 1713 F 107 CJST CJ 1795 1822 R 461 glyA 1054 CJST CJ 2060 2090 F 109 CJST CJ 2148 2174 R 463 pgm 1049 CJST CJ 2636 2668 F 113 CJST CJ 2753 2777 R 467 tkt 1048 CJST CJ 360 394 F 119 CJST CJ 442 476 R 472 aspA 1047 CJST CJ 584 616 F 121 CJST CJ 663 692 R 474 glnA [0133] The primers were used to amplify nucleic acid from 50 food product samples provided by s the USDA, 25 of which contained Campylobacterjejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacterjejuni clonal complexes and for distinguishing Campylobacterjejuni from Campylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates to demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 12A-C. Table 12A - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill down MLST Primer Pair Nos: 1048 and 1047 Base Ease MLST type or composition of Composition of or Cal Bioagent Bioagoent Group Species Isolate Complex by Cope by Strain Identifying Identifying origin Base SeSten Amplicon Amplicon Composition aequence Obtained with Obtained with Cn tisanalysis Primer Pair No: Primer Pair 1048 (aspA) No. 1047 (glnA) J-1 C. Goose ST 690 J ejuni /692/707/991 ST 991 RM3673 A30 G25 C16 T46 A47 G21 C16 T25 J-2 HC. Complex ST 356, jejun Human 206/4/353 complex RM4192 A30 G25 C16 T46 A48 G21 C17 T23 353 ________ J-2 Human Complex ST 436 RM4194 A30 G25 C15 T47 A48 G21 C18 T22 I___ Jelunl _____ 3541179 1_____ ____________ ST 257, J-4 Human Complex 257 complex RM4197 A30 G25 C16 T46 A48 G21 C18 T22 jejunl 257 J-5 Human Complex 52 ST 52 R4277 A30 G25 C16 T46 A46 G21 C17 T23 ST 51, RM4275 A30 G25 C15 T47 A48 G21 C17 T23 J-6 C. Human Complex 443 complex fejuni 443 RM4279 A30 G25 C15 T47 A48 G21 C17 T23 J-7 C. Human Complex 42 S 42 RM1864 A30 G25 C15 T47 A48 G21 CIO T22 jejunl ua omlx4 complex 42 n Complex ST 362, J-8 C. Human962 complex RH3193 A30 G25 C15 T47 A48 G21 CI T22 J-9 Human Complex ST 147, M3203 A30 G25 C15 T47 A47 G21 C18 T23 jejuni 45/263 Complex 45 C____ ejn Human Consistent ST 628 8RM4163 A.31 G27 C20 739 A.46 G21 C16 124 -67 with 74 ST 832 RM1169 A31 G27 C20 T39 A48 G21 C16 T24 closely related ST 1056 RM1857 AL31 G27 C20 T39 A48 G21 C16 T24 sequence types (none ST 889 RM1166 A31 G27 C20 T39 A48 G21 C16 T24 belong to a clonal ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24 complex) ST 1050 RM1518 A31 G27 C20 T39 A48 G21 C16 T24 ST 1051 RM1521 A31 G27 C20 T39 A48 G21 C16 T24 ST 1053 RM1523 A31 G27 C20 T39 A48 G21 C16 T24 ST 1055 RM1527 A31 G27 C20 T39 A48 G21 C16 T24 Poultry ST 1017 RM1529 A31 G27 C20 T39 A48 G21 C16 T24 C-1 C. coll ST 860 RM1840 A31 G27 C20 T39 A48 G21 C16 T24 ST 1063 RM2219 A31 G27 C20 T39 A48 G21 C16 T24 ST 1066 RM2241 A31 G27 C20 T39 A48 G21 C16 T24 ST 1067 RM2243 A31 G27 C20 T39 A48 G21 C16 T24 ST 1068 RM2439 A31 G27 C20 T39 A48 G21 C16 T24 ST 1016 RM3230 A31 G27 C20 T39 A48 G21 C16 T24 Swine ST 1069 RM3231 A3I G27 C20 T39 A48 G21 C16 T24 ST 1061 RM1904 A31 G27 C20 T39 A48 G21 C16 T24 ST 825 RH1534 A31 G27 C20 T39 A48 G21 C16 T24 Unknown ST 901 RM1505 A31 G27 C20 T39 A48 G21 C16 T24 C-2 C. coll Human ST 895 ST 895 RM1532 A31 G27 C19 T40 A48 G21 C16 T24 Consistent ST 1064 RM2223 A31 G27 C20 T39 A48 G21 C16 T24 with 63 closely ST 1082 RM1178 A31 G27 C20 T39 A48 G21 C16 T24 Poultry related C-3 C. coll sequence ST 1054 RM1525 A31 G27 C20 T39 A48 G21 C16 T24 types (none belong to a ST 1049 RM1517 A31 G27 C20 T39 A48 G2'1 C16 T24 clonal Marmoset complex) ST 891 RM1531 A31 G27 C20 T39 IA48 G21 C16 T24 Table 12B - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill down MLST Primer Pair Nos: 1053 and 1064 Base Base MLST type or Composition of Composition of Clonal Bioagont Bioagent Gro species Isolate Complex by Coe Identifying Identifying origin Base mp by Strain Amplicon Amplicon Composition sequence Obtained with Obtained with analysis analysisPrmrai ia ar No: 1053 (gltA) No: 1064 (g1yA) un1 C. Goose S9/07 ST 991 RM3673 A24 G25 C23 T47 A40 G29 C29 T45 jejuni 1692/707/991 S 9 137 J-2mC. Complex ST 356, A24 G25 C23 T47 A40 G29 C29 T45 -2 njun 206/48/353 complex RM4192 jejuni353 J-3 Human Complex ST 436 RM4194 A24 G25 C23 T47 A40 G29 C29 T45 jejuni 354/179 ST 257, A24 G25 C23 T47 A40 G29 C29 T45 J-4 C Human Complex 257 complex RM4197 jejuni257 J-5 Human Complex 52 ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 leiunl complex 52 ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46 J7-6 Ca.i Human Complex 443 complex J-6 443 RM4279 A24 G25 C23 T47 A39 G30 C28 T46 A24 G25 C23 T47 A39 G30 C26 T48 -7 uni Human Complex 42 compe 42 R - 68 ST 362, 24 G25 C23 T47 A38 G31 C28 T46 jun Human C x62 Complex RM3193 A24 G25 C23 T47 A38 G31 C28 T46 J-9 Human Complex ST 147, RM3203 jejunl 45/283 Complex 45 C8 A23 G24 C26 T46 A39 G30 C27 T47 jejunlST 826 RM4183 uAST 832 RM1169 A23 G24 C26 T46 A39 G30 C27 T47 ST 1056 RM1857 A23 G24 C26 T46 A39 G30 C27 T47 ST 889 RH1166 A23 G24 C26 T46 A39 G30 C27 T47 ST 829 RM1182 A23 G24 C26 T46 A39 G30 C27 T47 ST 1050 RM1518 A23 G24 C26 T46 A39 G30 C27 T47 ST 1051 RM1521 A23 G24 C26 T46 A39 G30 C27 T47 ST 1053 RM1523 A23 G24 C26 T46 A39 G30 C27 T47 Consistent with 74 ST 1055 RM1527 A23 G24 C26 T46 A39 G30 C27 T47 Poultry closely related ST 1017 RM1529 A23 G24 C26 T46 A39 G30 C27 T47 sequence C-1 C. coll types (none ST 860 RM1840 A23 G24 C26 T46 A39 G30 C27 T47 belong to a clonal ST 1063 RM2219 A23 G24 C26 T46 A39 G30 C27 T47 complex) ST 1066 RM2241 A23 G24 C26 T46 A39 G30 C27 T47 ST 1067 RM2243 A23 G24 C26 T46 A39 G30 C27 T47 ST 1068 RM2439 A23 G24 C26 T46 A39 G30 C27 T47 ST 1016 RM3230 A23 G24 C26 T46 A39 G30 C27 T47 Swine ST 1069 RM3231 A23 G24 C26 T46 NO DATA ST 1061 RM1904 A23 G24 C26 T46 A39 G30 C27 T47 ST 825 RM1534 IA23 G24 C26 T46 A39 G30 C27 T47 ST 901 RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. coll Human ST 895 ST 895 RM1532 A23 G24 C26 T46 A39 G30 C27 T47 Consistent ST 1064 RM2223 A23 G24 C26 T46 A39 G30 C27 T47 with 63 closely ST 1082 -RM1178 A23 G24 C26 T46 A39 G30 C27 T47 C-3 C. call rly uen e ST 1054 RM1525 A23 G24 C25 T47 A39 G30 C27 T47 types (none belong to a ST 1049 RM1517 A23 G24 C26 T46 A39 G30 C27 T47 clonal Marmoset complex) ST 891 R11531 A23 024 C26 T46 A39 030 C27 T47 Table 12C - Results of Base Composition Analysis of 50 Campylobacter Samples with Drill down MLST Primer Pair Nos: 1054 and 1049 BasQ Base MLST type or M Composition of Composition of Clonal Clonal Bioagent Bioagent Gro Spcies l Complex by Come bydentifyng Identifying origin Base SeqCompe Amplicon Amplicon Composition an e Obtained with Obtained with Compsit analysis Primer Pair No: Primer Pair 1 _ 1054 (pgm) No: 1049 (tkt) 1 Gue 69707/991 ST 991 RM3673 A26 G33 C18 T38 A41 G28 C35 T38 C.un Complex ST 356, -2 Human 0 353 complex RM4192 A26 G33 C19 T37 A41 G28 C36 T37 -3 Human8/35 S43 R49353 J3 C. ui Human Complex ST 436 RM4194 _____jJil ______ 354/179 P.27 G32 C19 T37 P.42 028 C36 T36 -69 ST 257, C- Human Complex 257 plex RM4197 A27 G32 C19 T37 A41 G29 C35 T37 -4 jejun 257 juni Human Complex 52 cmlex 52 RM4277 A26 G33 C18 T38 A41 G28 C36 T37 C. ST 51, RM4275 A27 G31 C19 T38 A41 G28 C36 T37 j-6 Human Complex 443 complex ______ _443 RM4279 A27 G31 C19 T38 A41 G28 C36 737 j-7 3 i Human Complex 42 co 604, RM1864 A27 G32 C19 T37 A42 G28 C35 T37 jejun!i Hmn Cmlx4 complex 42 Huma ComlexST 362, C- 82/4/36 complex RM3193 A26 G33 C19 T37 A42 G28 C35 T37 J-8___ _______uman_4/49/36 362 J-9 uni Human 4omlex 4 RM3203 A28 G31 C19 T37 A43 G28 C36 T35 C. ST 820 RM.4183 jejnlS 828_RM13 A27 G30 C19 T39 A46 G28 C32 T36 Human ST 832 RM1169 A27 G30 C19 T39 A46 G28 C32 T36 ST 1056 RM1857 A27 G30 C19 T39 A46 G28 C32 T36 ST 889 RM1166 A27 G30 C19 T39 A46 G28 C32 T36 ST 829 RM1182 A27 G30 C19 T39 A46 G28 C32 T36 ST 1050 RM1518 A27 G30 C19 T39 A46 G28 C32 T36 ST 1051 RM1521 A27 G30 C19 T39 A46 G28 C32 T36' ST 1053 RM1523 A27 G30 C19 T39 A46 G28 C32 T36 Consistent ST 1055 RM1521 with 74 A27 G30 C19 T39 A46 G28 C32 T36 Poultry closely acted ST 1017 RM1529 A27 G30 C19 T39 A46 G28 C32 T36 C-1 C. Collf tye (one ST 860 RM1840 A2G3C1T9 A4G8C2T6 blex) ST 103a21 A27 G30 C19 T39 A46 G28 C32 T36 ST 1063 RM2241 STmplex 1A27 G30 C19 T39 A46 G28 C32 T36 ST 1068 RM2243 A27 G30 C19 T39 A46 G28 C32 T36 ST 1076 RM323 A27 G30 C19 T39 A46 G28 C32 T36 SwltedST 1069 R3243 A27 G30 C19 T39 A46 G28 C32 T36 ST 1016 RM1304 A27 G30 C19 T39 A46 G28 C32 T36 Swin ST co69 ty32e1 Anon ST0 C60 T39 A4 G834T3 Seon to6 aM90 I_________ A27 030 019 T39 A46 028 C32 736 Unknown ST 825 RM1534 A27 G30 C19 T39 A46 G28 C32 T36 ST 901 RM1505 A27 G30 C19 T39 A46 G28 C32 T36 C-2 C. Coll Human ST 895 ST 895 RM1532 A27 G30 C19 T39 A45 G29 C32 T36 Consistent ST 1064 RM2223 A27 G30 C19 T39 A45 G29 C32 T36 ely ST 1082 RM1170 A27 G30 C19 T39 A45 G29 C32 T36 Poultry related C-3 C. Col n sequence ST 1054 RM1525 A27 030 019 739 A45 028 032 T36 types (noneA2G3C1T3 IA4G9C2T6 belong to a ST 1049 RM1517 n clonal A27 G30 C19 T39 A45 G29 C32 T36 M-r0oet complex) ST 891 RM1531 A27 G30 C19 T39 A45 G29 C32 T36 [0134] The base composition analysis method was successful in identification of 12 different strain groups. Campylobacterjejuni and Campylobacter coli are generally differentiated by all - 70 loci. Ten clearly differentiated Campylobacterjejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacterjejuni. One isolate (RM4183) which was designated as Campylobacterjejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli S by full MLST sequencing. [0135] Example 12: Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance [0136] To test the capability of the broad range survey and division-wide primer sets of Table 4 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners). In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship. [01371 Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346 349, 360, 361, 354, 362 and 363 (Table 4) all produced bacterial bioagent amplicons which identified A cinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae 0 was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from I to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively. as [01381 The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait). The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 30 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIR as a result of the spread of strain 7 from the index patient.

- 71 [0139] The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated. Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may 5 provide evidence that at least some of the infections at that locale were a result of a nosocomial infections. [01401 This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for to epidemiological surveillance. [0141] Example 13: Selection and Use of MLST Acinetobacter baunanii Drill-down Primers [0142] To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by multi 15 locus sequence typing (MLST) such as the MLST methods of the MLST Databases at the Max Planck Institute for Infectious Biology (web.mpiib-berlin.mpg.de/mlst/dbs/Mcatarrhalis/ documents/primersCatarrhalishtml), an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down MLST analogue primers hybridize for production of bacterial bioagent identifying amplicons ao include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 13. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and Q s amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. The primer names given in Table 13 indicates the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named 30 ABMLST-1 1-OIF007_62_91_F because it hybridizes to the Acinetobacter MLST primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91.

-72 Table 13: MLST Drill-Down Primers for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter Primer Forward Primer Name Forward Reverse Primer Name Reverse pair Primer Primer No. (SEQ ID NO:) (SEQ ID NO:) 1151 AB MLST-11-OIF007 62 91 F 83 AB MLST-11-OIF007 169 203 R 426 1152 AB MLST-11-OIF007 185 214 F 76 AB MLST-11-OIF007 291 324 R 432 1153 AB KLST-11-OIF007 260 289 F 79 AB MLST-11-OIF007 364 393 R 434 1154 AB MLST-11-OIF007 206 239 F 78 AB MLST-11-OIF007 318 344 R 433 1155 AR MLST-11-OIF007 522 552 F 80 AB MLST-11-01F007 587 610 R 435 1156 AB MLST-11-OIF007 547 571 F 81 AB MLST-11-OIF007 656 686 R 436 1157 AB MLST-11-OIF007 601 627 F 82 AB MLST-11-OIF007 710 736 R 437 1158 AB_MLST-11- 65 O1F007 1202 1225 F AB MLST-11-01F007 1266 1296 R 420 1159 ABMLST-11- 65 O1F007 1202 1225 F AB MLST-11-OIF007 1299 1316 R 421 1160 ABMLST-11- 66 OIF007 1234 1264 F AB MLST-11-OIF007 1335 1362 R 422 1161 AR_MLST-11- 67 OIF007 1327 1356 F AB MLST-11-OIF007 1422 1448 R 423 1162 ABMLST-11- 68 0IF007 1345 1369 F AB MLST-11-OIF007 1470 1494 R 424 1163 ABMLST-11- 69 OIF007 1351 1375 F AB MLST-11-01F007 1470 1494 R 424 1164 ABMLST-11- 70 01F007 1387 1412 F AB MLST-11-01F007 1470 1494 R 424 1165 ABMLST-11- 71 OIF007 1542 1569 F AB MLST-11-OIF007 1656 1680 R 425 1166 AB_MLST-11- 72 OIF007 1566 1593 F AB MLST-11-01F007 1656 1680 R 425 1167 ABKLST-11- 73 OIF007 1611 1638 F AB MLST-11-01F007 1731 1757 R 427 1168 ABMLST-11- 74 OIF007 1726 1752 F AB MLST-11-OIF007 1790 1821 R 428 1169 AB_MLST-11- 75 OIF007 1792 1826 F AB MLST-11-OIF007 1876 1909 R 429 1170 ABMLST-11- 75 OIF007 1792 1826 F AB MLST-11-OIF007 1895 1927 R 430 1171 AB MLST-11- 77 AB MLST-11-OIF007 2097 2118 R 431 -73 01F007 1970 2002 F [0143] Analysis of bioagent identifying amplicons obtained using the primers of Table 13 for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (STI 1) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 2 8 " 5 Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage. [0144] All of the sample isolates were tested against a broad panel of antibiotics to characterize 10 their antibiotic resistance profiles. As 'n example of a representative result from antibiotic susceptibility testing, STI I was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbipenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics. S Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a o time-scale previously not achievable. [0145] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not Zs limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims (19)

1. A composition comprising an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 350. 5

2. The composition of claim 1, wherein said oligonucleotide primer comprises at least one modified nucleobase.

3. The composition of claim I or 2, further comprising a second oligonucleotide 10 primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 712.

4. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one modified nucleobase. 15

5. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the 5'-end.

6. The composition of claim 3 wherein either or both of said oligonucleotide 20 primers comprises at least one non-template tag.

7. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one molecular mass modifying tag. 25

8. A kit comprising the composition of claim 3.

9. The kit of claim 8 further comprising at least one calibration polynucleotide.

10. The kit of claim 8 further comprising at least one ion exchange resin linked to 30 magnetic beads.

11. A method for identification of an unknown bacterium comprising: amplifying nucleic acid from said bacterium using the composition of any one of claims 3 to 7 to obtain an amplification product; 75 determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification s product with a plurality of molecular masses or base compositions of known bacterial bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of a member of said plurality of molecular masses or base compositions identifies said unknown bacterium. 10

12. The method of claim 11 wherein said molecular mass is determined by mass spectrometry.

13. A method of determining the presence or absence of a bacterium of a is particular clade, genus, species, or sub-species in a sample comprising: amplifying nucleic acid from said sample using the composition of any one of claims 3 to 7 to obtain an amplification product; determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product 20 from said molecular mass; and comparing said molecular mass or base composition of said amplification product with the known molecular masses or base compositions of one or more known clade, genus, species, or sub-species bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the 25 molecular mass or base composition of one or more known clade, genus, species, or sub species bioagent identifying amplicons indicates the presence of said clade, genus, species, or sub-species in said sample.

14. The method of claim 13 wherein said molecular mass is determined by mass 30 spectrometry.

15. A method for determination of the quantity of an unknown bacterium in a sample comprising: 76 contacting said sample with the composition of any one of claims 3 to 7 and a known quantity of a calibration polynucleotide comprising a calibration sequence; concurrently amplifying nucleic acid from said bacterium in said sample with the composition of any one of claims 3 to 7 and amplifying nucleic acid from said 5 calibration polynucleotide in said sample with the composition of any one of claims 3 to 7 to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon; determining the molecular mass and abundance for said bacterial bioagent identifying amplicon and said calibration amplicon; and 1o distinguishing said bacterial bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in said sample. is

16. The method of claim 15 further comprising determining the base composition of said bacterial bioagent identifying amplicon.

17. A method for identification of an unknown bacterium, said method according to claim 11 and substantially as hereinbefore described with reference to any one of the 20 examples.

18. A method of determining the presence or absence of a bacterium, said method according to claim 13 and substantially as hereinbefore described with reference to any one of the examples. 25

19. A method for determination of the quantity of an unknown bacterium, said method according to claim 15 and substantially as hereinbefore described with reference to any one of the examples. 30 Dated 7 March, 2011 Ibis Biosciences, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON