KR100812795B1 - Primer and probe for detection of bacillus anthracis and method for detecting bacillus anthracis using thereof - Google Patents

Abstract

A method for detecting Bacillus anthracis is provided to detect the Bacillus anthracis specifically and distinguish B. cereus, which is very similar to the Bacillus anthracis taxonomically and molecular biologically, from the Bacillus anthracis. A probe for detecting Bacillus anthracis is at least one selected from the group consisting of: (a) a sequence coding an amino acid sequence of SEQ ID : NO. 2; (b) a sequence complimentary to the sequence of the (a); and (c) a consecutive sequence more than 20bp which is derived from the sequence of the (a) or (b) and includes a sequence selected from the group consisting of sequences of SEQ ID : NOs. 3 to 44. A primer for detecting Bacillus anthracis includes a sequence selected from the group consisting of sequences of SEQ ID : NOs. 3 to 44. To detect Bacillus anthracis, at least one of the probe and the primer is used through PCR(Polymerase Chain Reaction), DNA sequencing, hybridization by microarray, PCR-RFLP(restriction fragment length polymorphism), RAPD(randomly amplified polymorphic DNA), DAF(DNA amplification fingerprinting), AP-PCR(arbitrarily primed PCR), STS(sequence tagged site), EST(expressed sequence tag), SCAR(sequence characterized amplified regions), ISSR(inter-simple sequence repeat amplication), AFLP(amplified fragment length polymorphism), CAPS(cleaved amplified polymorphic sequence), PCR-SSCP(single-strand conformation polymorphism), Northern hybridization or Southern hybridization. Further, the probe is accumulated on a substrate.

Description

Primer and probe for detection of Bacillus anthracis and method for detecting Bacillus anthracis using approximately}

1 is Bacillus Schematic (Suppression subtractive hybridization) with the genomic DNA of B. cereus and B. anthracis .

2A is a Bacillus to connect the adapter Cereus (B.cereus) and Bacillus anthracis, and to process the results of genomic DNA with Rsa I of (B. anthracis), diagram B of Figure 2 SSH (Suppression subtractive hybridization) are four, and the result of amplification by nested PCR, FIG. C of 2 is a result of PCR amplification of the plasmid confirmed cloning for reverse Southern hybridization.

FIG. 3 shows the results of reverse southern hybridization to select B. anthracis specific clones.

A: ECL labeled Bacillus Cereus When using genomic DNA as a probe

B: ECL labeled anthrax genomic DNA was used as a probe

Figure 4 shows the strain for each strain (strain) used as a template (template) when performing PCR using the primer of the present invention.

5 and 6 are photographs showing the results of PCR analysis using primer combinations of SEQ ID NO: 43 and SEQ ID NO: 44.

Lanes 1 to 8: B. anthracis genomic DNA was used as a template

Lane 9 ~ Lane 78: Genetic DNA of Bacillus strains other than Anthrax was used as a template

The present invention relates to a primer for detecting anthrax, a probe and a method for detecting anthrax using the same, more specifically, using a gene encoding a glycosyl transferase consisting of amino acids of SEQ ID NO: 2 as a target for detection. The present invention relates to anthrax glycosyl transferase gene and primers derived from complementary sequences, probes, and methods for detecting anthrax using the same.

Anthrax is Bacillus Anthracis ) is an infectious disease characterized by an increase in the spleen, swelling and bleeding of the subcutaneous mucosa. Anthrax Bacillus anthracis) is on the basis of morphological and DNA affinity, Bacillus Cereus (B. cereus), Basiliscus written Lindsay N-Sys (B. thuringiensis), Bacillus Edith M. (B. mycoides), Bacillus by Henderson seutepa norbornene during scan (B. weihenstephanensis) and Bacillus With Pseudomonas Edith M. (B. pseudomycoides) Bacillus cereus (B. cereus) belongs to a group (Lechner et al, Int J. Syst Bacteriol 48:..... 1373-1382, 1998; Nakamura, Int J. Syst Bacteriol. 48: 1031-1035, 1998).

Once anthrax has a high mortality rate within a short time, rapid and accurate diagnosis is required. Moreover, as anthrax is bioinorganized, an appropriate biomarker for rapid screening is needed. Anthrax ( B. anthracis ) is generally identified by five characteristics, including capsular positivity, non-motile, non-hemorrhagic, penicillin sensitivity, and pathogenicity to mice (Choi et al., Korean Journal of Microbiology 27: 93-102, 1992). recently, anthrax, and the appearance is also typical biological properties and mutant anthrax visible differences (B. anthracis) strains (B. anthracis) difficulty in identifying Bacillus also commonly isolated from clinical specimen Edith M. (B. mycoides) and Bacillus Cereus (B. cereus) culture and even biological properties are often incorrectly identified as Bacillus anthracis (B. anthracis) tests in the lab because of the similarity with Bacillus anthracis (B. anthracis) (, microbes, etc. for Kim Journal 34: 311-319, 1999: Kim et al., Korean Journal of Microbiology 35: 1-10, 2000).

Thus, Bacillus Cereus Many studies have been conducted to identify and identify B. anthracis from group species. Anthrax is characterized by a large colony (Choi et al., Korean Journal of Microbiology 27: 93-102, 1992), Ochterlony immunodiffusion test using immunoserum (Choi et al., Korean Society for Microbiology 27: 407-417. 1992) , Specific antigen detection using monoclonal antibodies (Choi et al., Korean Journal of Microbiology 29: 535-545, 1994; Ezzell et al., J. Clin. Microbiol. 28: 223-231, 1990), Microhemagglutination (Buchanan) et al., J. Immunol. 107: 1631-1636, 1971), enzyme immunoassay (Johnson-Winegar, J. Clin. Microbiol. 20: 357-361, 1984; Kleine-Albers et al., Infect. 35: 235-239, 1994), isolation of pXO1 and pXO2 plasmids encoding exotoxins (Mikesell et al., Infect. Immun. 39: 371-376.1983), API testing (Logan et al., J. Med. Microbiol. 20: 75-85.1985). However, the methods described above are time consuming, and due to the complexity of the techniques, it is difficult to be used for identification of general laboratories, and it is impossible to identify genetic correlations between the strains.

In order to overcome the above problem, there have been many efforts to use a rapid detection method based on DNA such as polymerase chain reaction (PCR). However, PCR methods that have been developed for the detection of anthrax chromosomes are Ba813 (Patra et al., FEMS Immunol. Med. Microbiol. 15: 223-231), vrrA gene (Jackson et al., Appl. Environ. Microbiol. 63: 1400-1405), gyrB gene (Yamada et al., Appl. Environ. Microbiol. 65: 1483-1490), SG-850 (Daffonchio et al., Appl. Environ. Microbiol. 65: 1298-1303), rpo B Gene (Qi et al., Appl. Environ. Microbiol. 65: 1483-1490) genes, etc., the false positive results in the detection of anthrax chromosomes, the problem of lack of specificity has emerged.

In particular, Bacillus anthracis (B. anthracis) is classified to the closest haksang Bacillus celebrity's right (B. cereus) and 16S rRNA as compared to the difference of the nucleotide sequence does not, only 23S rRNA change in a single nucleotide base in a nucleotide sequence or inserting only Because it is present (Ash & Collins, FEMS Microbiol. Lett. 94: 75-80, 1992; Ash et al., Int. J. Syst. Bacteriol. 41: 343-346, 1991) and B. anthracis Bacillus Cereus was a distinct limit on (B. cereus).

Thus, strains belonging to the genus Bacillus , in particular Bacillus There is an urgent need to develop a method for distinguishing cereus from anthrax.

Accordingly, the present inventors prepared primers and probes by using a gene encoding a glycosyl transferase having an amino acid of SEQ ID NO: 2 as a target for detection, and the primers and probes can specifically detect only anthrax. The present invention has been completed by confirming that it can.

Accordingly, an object of the present invention is to provide anthrax glycosyl transferase gene and complementary base sequence derived from anthrax glycosyl transferase gene, which is designed to effectively detect anthrax by using the glycosyl transferase gene of anthrax as a target of detection. It is to provide a primer, a probe and a method for detecting anthrax using the same.

In order to achieve the object of the present invention as described above, the present invention comprises a gene encoding a glycosyl transferase having a amino acid of SEQ ID NO: 2, a base sequence complementary thereto and a base sequence derived therefrom It provides a probe and microarray for anthrax detection characterized in that the probe is integrated on a substrate.

The present invention also provides a primer having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 derived from a gene encoding a glycosyl transferase having an amino acid of SEQ ID NO: 2.

The present invention also provides a method for detecting anthrax using the primer or probe.

The present invention also provides a composition for detecting anthrax, including the primer or probe.

Hereinafter, the present invention will be described in detail.

The present invention provides a probe for anthrax detection. Probes provided in the present invention (a) a base sequence encoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence complementary to (a) above; And (c) a sequence selected from the group consisting of 20 bp or more consecutive nucleotide sequences derived from the nucleotide sequences of (a) or (b) and comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44; Can have Preferably, the base sequence of (c) is 20bp or more and 50bp or less, derived from the nucleotide sequence of (a) or (b) and comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 Preferably, the sequence is 20 bp or more and 100 bp or less. In addition, the base sequence of (a) is preferably composed of the base sequence described in SEQ ID NO: 1. In addition, the probe of the present invention may be modified (eg, added, deleted, substituted) within a range that does not affect anthrax specific detection.

The gene encoding the glycosyl transferase having the amino acid sequence of SEQ ID NO: 2 is characterized by anthrax specific. Therefore, the probe of the present invention consists of a gene encoding a glycosyl transferase having the amino acid sequence of SEQ ID NO: 2, a nucleotide sequence complementary thereto or a nucleotide sequence derived therefrom, which is specific for anthrax and in recent years. Since Bacillus spp. Strains having a high relationship have non-specific characteristics, anthrax can be efficiently detected. In one embodiment of the present invention, it was confirmed that the glycosyl transferase gene of anthrax is a specific gene that can distinguish anthrax from a strain of the genus Bacillus having high relationship (Example 1).

The present invention provides a gene encoding a glycosyl transferase having an amino acid of SEQ ID NO: 2 and a probe derived from a base sequence complementary thereto, and the probe is integrated on a substrate. Provide an array.

In the present invention, the term “probe” refers to a nucleic acid fragment corresponding to a few to several hundreds of bases that can achieve specific binding with DNA or RNA, and is labeled to confirm the presence or absence of a specific DNA or RNA. have. Probes can be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like, and are labeled with biotin, FITC, rhodamine, DIG, etc. Or radiolabeled isotopes.

The present invention also provides a microarray for detecting anthrax, wherein the probe is integrated on a substrate.

The microarray may include DNA or RNA polynucleotides. The microarray consists of conventional microarrays except that the polynucleotide of the present invention is included in the probe polynucleotide.

Methods of preparing microarrays by immobilizing probe polynucleotides on substrates are well known in the art. The term “probe polynucleotide” refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid.

The process of immobilizing probe polynucleotides associated with anthrax of the present invention on a substrate can also be readily prepared using this prior art. In addition, the hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is accomplished by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising, for example, a fluorescent substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating from the labeling substance. The hybridization result can be detected by detecting the signal.

The present invention also provides a primer for anthrax detection.

Preferably, the primer provided in the present invention may have a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44. In addition, the primers of the present invention may be modified (eg, added, deleted, substituted) within a range that does not affect anthrax specific detection. The primer of the present invention is a sequence derived from a gene consisting of a nucleotide sequence encoding a glycosyl transferase having an amino acid sequence of anthrax specific SEQ ID NO: 2 is selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 It consists of a base sequence. In an embodiment of the present invention, PCR was performed using a primer combination of SEQ ID NO: 43 and SEQ ID NO: 44. As a result, anthrax and other Bacillus strains (particularly, Bacillus cereus ( B. cereus )) can be distinguished (see FIGS. 5 and 6).

As used herein, the term “primer” refers to a short nucleic acid sequence that can form base pairs with complementary templates and that serves as a starting point for template strand copying.

The primers may include restriction fragment length polymerphism (RFLP), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site (STS), expressed sequence tag (EST), SCAR (sequence characterized amplified regions), inter-simple sequence repeat amplication (ISSR), amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence (CAPS), single-strand conformation polymorphism (PCR-SSCP), and the like. It can be designed to suit a variety of DNA analysis.

The present invention also (a) a base sequence encoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence complementary to (a) above; And (c) at least one probe selected from the group consisting of 20 bp or more consecutive nucleotide sequences derived from the nucleotide sequences of (a) or (b) and comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 Or it provides a method for detecting anthrax using a probe having a base sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44.

As a method for detecting anthrax using the primers or probes of the present invention, PCR (Polymerase Chain Reaction) analysis, DNA sequencing, hybridization by microarray, PCR-RFLP (restriction fragment length) polymerphism analysis, randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site (STS), expressed sequence tag (EST), sequence characterized amplified regions (SCAR), ISSR (inter-simple sequence repeat amplication), AFLP (amplified fragment length polymorphism), CAPS (cleaved amplified polymorphic sequence), PCR-SSCP (single-strand conformation polymorphism), Northern hybridization (Northern hybridization) Southern hybridization ( But may be performed using various DNA polymorphism assays known in the art such as Southern hybridization and Western hybridization. It is not limited.

Specifically, in an embodiment of the present invention, a method of specifically detecting anthrax by PCR using anthrax glycosyl transferase gene and a primer derived from a base sequence complementary thereto is illustrated. That is, PCR analysis was performed using a primer combination of SEQ ID NO: 43 and SEQ ID NO: 44. As a result, it was confirmed that the primer of the present invention can specifically detect anthrax by specifically amplifying only the glycosyl transferase gene of anthrax (see FIGS. 5 and 6).

The present invention also (a) a base sequence encoding the amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence complementary to (a) above; And (c) at least one probe selected from the group consisting of 20 bp or more consecutive nucleotide sequences derived from the nucleotide sequences of (a) or (b) and comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 Or it provides a composition for detecting anthrax, including a probe having a base sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44.

The composition may include a variety of reagents, such as PCR reaction mixtures, restriction enzymes, agarose, hybridization, and the like, for example, an experimental procedure (eg, PCR, Southern blot, etc.) necessary for detecting anthrax by using the primer or probe. And / or buffers required for electrophoresis.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention to this embodiment.

< Example  1>

SSH ( Suppression subtractive hybridization Of anthrax-specific genomes

<1-1> Genomic DNA ( Genomic DNA ) detach

Isolation of genomic DNA is anthrax (B. anthracisChangnyeong, Korea (obtained from Veterinary Science and Quarantine Service, July 6, 2000)Bacillus Cereus(B. cereusKCTC 3624T was used as an experimental strain, and was performed according to Ausubel et al. (Ausubel et al., Current protocols in molecular biology, section 22.4, John Wiely and Sons, New York, N.Y., 1994).

First, the experimental strains incubated in 3 ml of nutrient broth (Difco) were transferred to 1.5 ml microtubes and centrifuged at 15,000 rpm for 2 minutes to precipitate the cells. The precipitated cells were well suspended by adding 100 µl of TE buffer (pH 8.0), and 20 µl of 10% sodium dodecyl sulfate (SDS) and 10 µl of proteinase K (10 mg / ml) at 50 ° C. The reaction was carried out for 1 hour. The reacted cells were mixed well by adding 200 μl of 10% CTAB (cetyltrimethylammonium bromide) /0.7 M NaCl solution, followed by reaction at 65 ° C. for 10 minutes, and the same amount of chloroform / isoamyl alcohol (24: 1) solution was added thereto. After shaking for 5 minutes and centrifuged for 5 minutes at 4 ℃, only the supernatant was transferred to a new tube. Genomic DNA was precipitated by adding 1/10 times 3M sodium acetate and 2 times 100% ethanol. The precipitated genomic DNA was washed with 70% ethanol, dissolved in 50 μl of TE buffer, RNase was added to a final concentration of 20 μg / ml, and then reacted at 42 ° C. for 30 minutes to remove RNA. The purified DNA was used for the experiment while quantifying at 260nm using a spectrometer (Model MBA 2000, Perkin-Elmer, Norwalk, CN, USA) and stored at -20 ℃.

<1-2> restriction enzyme cleavage

In order to connect an adapter to the genomic DNA, the extracted genomic DNARsaI restriction enzyme was treated.

Example <1-1> Purification by anthrax in (B. anthracis) Korea main CR and Bacillus cereus (B. cereus) to genomic DNA restriction enzyme for each of the 2 ㎍ KCTC 3624T Rsa I (10 unit / ㎕, Roche Molecular Biochemicals, Mannheim, Germany) 1.5 μl and 5 μl of 10X buffer were added for 5-16 hours in a 37 ° C. constant temperature water bath, and then 100 V on 1.0% LE agarose gel (FMC Bioproducts, Rockland, ME, USA). Electrophoresis was performed for 20 minutes. After electrophoresis, staining was performed with EtBr (ethidium bromide) (10 mg / ml) for 10 minutes, and the presence or absence of cleavage was checked using a Gel Doc 2000 system (Bio-Rad, Hercules, CA, USA). As a result, as shown in FIG. 2A, it was confirmed that the cleavage was confirmed. After confirming the cleavage, 2.5 μl of 0.2 M EDTA solution was added to stop the reaction. The cleaved DNA was purified using a solution of phenol / chloroform / isoamyl alcohol (25: 24: 1), precipitated by adding 0.5 times of 3 M sodium acetate and 2.5 times of 100% ethanol, washed with 80% ethanol and 6.5 It was dissolved in 3 μl of sterile distilled water.

<1-3> SSH ( Suppressive Subtractive Hybridization )

Suppressive subtractive hybridization (SSH) used a hybridization kit (PCR-Select Bacterial Genome Subtractive Hybridization kit, Catalog #: K1809-1) from CLONTECH Laboratories (Palo Alto, CA USA). Shown in

First, in order to make adapter-ligated tester DNA ( B. anthracis ) of Changnyeong, Korea, the test strain, 1.8 μl of distilled water was added to 1.2 μl of B. anthracis genomic DNA digested with Rsa I. After dilution, 2 μl of 5 × ligation buffer, 1 μl of T4 DNA ligation (400 units / μl) and 1.8 μl of distilled water were added to prepare a ligation master mix. The ligation reaction mixture is adjusted to a final volume of 10 μl by mixing 1 μl of diluted DNA, 2 μl of 10 uM adapter 1 or 2 μl of adapter 2R and 7 μl of ligation master mix into two tubes, respectively. The reaction was overnight at 16 ° C. in a water bath.

2 μl of B. cereus KCTC 3624T DNA, 1 μl of 1-adaptor-linked tester DNA and hybridization buffer, a comparative strain for primary hybridization The total volume was adjusted to 4 μl and then denatured at 98 ° C. for 1 minute and 30 seconds, and hybridization was performed at 63 ° C. for 1 hour and 30 minutes. A 2R-adaptor-linked tester DNA sample was also performed in the same manner as above.

Secondary hybridization is comparative strain Bacillus Cereus (B. cereus) and KCTC 3624T to the addition of 2X hybridization buffer 1 ㎕ the genomic DNA 1 ㎕ denaturing for 1 min 30 sec at 98 ℃ following primary hybridization reaction solution (1-adaptor-linked The first hybridization reaction solution using tester DNA and two first hybridization reaction solutions using 2R-adaptor-linked tester DNA) were mixed well and reacted overnight at 63 ° C. 200 μl of dilution buffer was added thereto to react at 63 ° C. for 7 minutes and stored at −20 ° C.

<1-4> Nested  PCR ( nested PCR )

Anthrax (B. anthracisNested PCR was performed to identify specific genomes. 1 μl of a PCR primer (adaptor sequence specific primer 5′-CTAATACGACTCACTATAGGGC-3 ′, SEQ ID NO: 45) to 1 μl of the hybridization reaction solution of Example <1-3>, 2.5 μl of 10X buffer, Taq DNA polymerase (Roche) G) 1 μl and 19.5 μl of distilled water were added and reacted at 72 ° C. for 2 minutes, followed by 30 minutes at 94 ° C., 30 seconds at 66 ° C., and 1.5 minutes at 72 ° C. in a DNA thermal cycler (Model 480, Perkin Elmer). Was carried out 25 times. Again, 1 μl of the PCR product and 39 μl of distilled water were mixed, followed by secondary PCR. As primers, nested primer 1 (5'-TCGAGCGGCCGCCCGGGCAGGT-3 ', SEQ ID NO: 46) and nested primer 2R (5'-AGCGTGGTCGCGGCCGAGGT-3', SEQ ID NO: 47) were used. , 12 minutes of reaction was carried out at 72 ° C. for 1.5 minutes. PCR amplification products were confirmed by electrophoresis on 1.2% LE agarose gel (FMC), the results are shown in B of FIG.

<1-5> Cloning

PCR amplification products were cloned into pCR 2.1 vectors using the TOPO TA cloning system (Invitrogen, Carlsbad, CA, U.S.A.)E. coli Transformed to TOP 10.

First, 1 µl (1 U) of DNA ligase, 1 µl of 10X buffer, 1 µl of 10 mM ATP, and 6 µl of DW were sequentially added to 1 µl (10 ng) of the PCR product of Example <1-4> at room temperature. Ligation was performed for 5 minutes. 1 μl of ligation solution was taken, mixed well with 50 μl of E. coli TOP 10 competent cells, left for 30 minutes on ice and subjected to heat shock at 42 ° C. for 30 seconds to introduce DNA into cells. Transformed E. coli The culture was shaken for 1 hour at 37 ° C. at 220 rpm, then LB plate medium containing Ampicillin (50 μg / ml) and 12 μl of X-gal (50 mg / ml) (Sigma-Aldrich, St. Louis, Mo, USA) and incubated for 16 hours at 37 ℃. In order to measure the presence of PCR amplification products, only white colonies were taken from the colonies grown after incubation, inoculated into 5 ml of LB broth, shake-cultured at 37 ° C. for 220 hours at 220 rpm, and then plasmid DNA purification kit (Plasmid DNA). The plasmid DNA was isolated using a purification kit, Promega, Madison, WI, USA). The isolated plasmid DNA was digested with restriction enzyme EcoR I (Roche) for 1 hour at 37 ° C and electrophoresed with 1.0% LE agarose gel (FMC) to observe the cloning of PCR amplification products.

<1-6> Reverse Southern hybridization Reverse southern hybridization )

From the plasmid DNA confirmed cloning using the nested primer 1 and nested primer 2R used in Example <1-4> anthrax (B. anthracis) DNA was amplified by PCR (FIG. 2C). 30 μl of the amplified DNA was denatured by heating at 100 ° C. for 5 minutes, and then left on ice for 5 minutes, and the total volume was 100 μl by adding the same amount of 20X SSC (USB Co., Cleveland, OH, U.S.A.). Southern blot was used ECL kit (ECL Direct Nucleic Acid Labeling and Detection System, Amersham Biosciences, Little Chafont, Buckinghamshire, UK). Hybond-N + membrane (Amersham Biosciences), which was previously settled on 6X SSC, was placed in a Bio-Dot microfiltration device (Bio-Dot Microfitration Apparatus, Bio-Rad), and 500 μl of distilled water was dispensed into each well to add membrane. Singer Dispense 50 μl of DNA solution into each well, wash with 2X SSC, remove the membrane and dry on Whatmann 3 MM paper, then use a Stratalinker® UV crosslinker (Stratagene, La Jolla, Calif., USA) to 3 at 1000 J. Crosslinking was performed for 30 minutes. After crosslinking, 6 ml of ECL hybridization buffer was added and prehybridized at 42 ° C. for 30 minutes. Then, the labeled probe was added and reacted overnight. The labeled probe is anthrax (B. anthracis200 μl of genomic DNA (100 μg / ml)RsaAfter digestion with I (Roche), the reaction was carried out at 37 ° C. for 1 hour, dissolved in 30 μl of distilled water, and denatured at 100 ° C. for 10 minutes. It was prepared by reaction. On the other hand, after the reaction, the membrane was taken out, washed once in 20% at 0.4% SDS, 0.5X SSC, and twice in 5 minutes at 2X SSC, dried, and added with an ECL detection reagent to obtain a film cassette (Eastman Kodak, Rochester). , NY, USA) and developed on an ECL film (Eastman Kodak), the results are shown in Figure 3B.

On the other hand, in order to confirm that the clone specific to the anthrax is non-specific to B. cereus , reverse Southern hybridization was performed in the same manner as above except that Bacillus cereus genomic DNA was used as a probe. The results are shown in FIG. 3A.

As a result of comparing A and B of FIG. 3, 32 clones specific to anthrax and nonspecific to Bacillus cereus were selected.

<1-7> Gene Sequencing

32 clones positive for anthrax by Southern hybridization in Example <1-6> using a BigDye Terminatior Cycle Sequencing Kit, Applied Biosystems, Foster City, CA, USA Gene sequencing was analyzed. Recombinant plasmids were prepared using a Promega DNA purification kit (Promega).E. coliAfter purification from pure, 8 μl of Bigdye terminator ready reaction mixture and 3.2 pM of sequencing primer (M13 or SP6) were added to 2 μl (50 ng) of plasmid DNA and the final volume was adjusted to 20 μl with distilled water. Next, a cycle sequencing PCR was performed in a GeneAmp ™ PCR System 2700 (Applied Biosystems), repeating 25 times at 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 4 minutes. 2 μl of 3M sodium acetate (pH 4.6) and 50 μl of 95% ethanol were added thereto, and the mixture was allowed to stand at room temperature for 15 minutes, followed by centrifugation at 15,000 rpm for 20 minutes to precipitate DNA, washed with 100 μl of 70% ethanol, and then dried. The dye was removed. Purified DNA was dissolved in 13 μl of template suppression reagent (TSR), heated at 95 ° C. for 2 minutes, and allowed to stand on ice for 5 minutes and then developed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

The gene sequences analyzed were searched using the US National BLAST (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/blast), multi-aligned with the gene sequences listed in GenBank and deterministic similarity The protein coding site was analyzed using ORF finder (NCBI).

As a result of analysis, the analyzed sequence was GenBank No. AE017334.2 (GI: 50082967) exhibited 100% homology with the nucleotide sequences of 5010707 to 5011879, and thus could be identified as the glycosyltransferase gene (SEQ ID NO: 1) of anthrax.

< Example  2>

Polymerase chain reaction  Anthrax using B. anthracis ) Specific gene detection

<2-1> anthrax specific Of primer  making

Anthrax (B. anthracis )Primer 3 (http://frodo.wi.mit.edu/cgi-bin/) based on glycosyl transferase gene sequence among gene sequences selected by SSH (suppressive subtractive hybridization) for specific gene detection Primers were designed using the primer3 / primer3_www.cgi program (Table 1). The designed primer was commissioned by Genotech Co., Ltd. (Taejon, Korea) on a 200 pM scale and purified by PAGE. The synthesized primers were diluted in 10 μM concentration and stored at -20 ° C and used for the experiment.

Forward direction primer SEQ ID NO: Reverse primer SEQ ID NO: CAGCGTAATGTTCGAAATGC 3 CGATTGTTGCAGATGTTGCT 4 CAGCGTAATGTTCGAAATGC 5 TGTTGCAGATGTTGCTGGTT 6 CAGCGTAATGTTCGAAATGC 7 TTGTTGCAGATGTTGCTGGT 8 CAGCGTAATGTTCGAAATGC 9 ACCGATTGTTGCAGATGTTG 10 TTCTCCCCTAGTCGATTTCGT 11 CGATTGTTGCAGATGTTGCT 12 TCGCCTTCATTTTTCGTTTC 13 CGTATCCATCCAAAAACACG 14 AAGGACTCCGGCCATAAATC 15 TCCATCCAAAAACACGAAAA 16 CAGCGTAATGTTCGAAATGC 17 GCAGATGTTGCTGGTTATGC 18 TCTCCCCTAGTCGATTTCGT 19 CGATTGTTGCAGATGTTGCT 20 AAGGACTCCGGCCATAAATC 21 CGTATCCATCCAAAAACACG 22 TTCGCCTTCATTTTTCGTTT 23 CGTATCCATCCAAAAACACG 24 TTTCGCCTTCATTTTTCGTT 25 CGTATCCATCCAAAAACACG 26 TCGCCTTCATTTTTCGTTTC 27 ACGTATCCATCCAAAAACACG 28 TTAAGGACTCCGGCCATAAA 29 TCCATCCAAAAACACGAAAA 30 TTCTCCCCTAGTCGATTTCGT 31 ACCGATTGTTGCAGATGTTG 32 AAGGACTCCGGCCATAAATC 33 ACGTATCCATCCAAAAACACG 34 TTCGCCTTCATTTTTCGTTT 35 ACGTATCCATCCAAAAACACG 36 TTTCGCCTTCATTTTTCGTT 37 ACGTATCCATCCAAAAACACG 38 TTAAGGACTCCGGCCATAAA 39 CGTATCCATCCAAAAACACG 40 TCCATTCTCCCCTAGTCGAT 41 CGATTGTTGCAGATGTTGCT 42 TCTTCAGTGACAAAACCACA 43 CAAGAAATCTTTTTCGAAGG 44

<2-2> polymerase chain reaction PCR )

The synthesized primer is anthrax (B. anthracisIn order to confirm that only) can be specifically detected, polymerase chain reaction (PCR) was performed using a primer combination of SEQ ID NO: 43 and SEQ ID NO: 44 among the synthesized primers. First, the separated and purified anthrax (see FIG. 4)B. anthracis) And 1 μl (10 ng) of DNA of control strains (2 μl 10X Taq buffer, 8 μl 2.5 mM dNTPs, 10 μl 1.25 mM MgCl 2, 59 μl DW, 1 μl 10 uM forward primer, 10 uM reverse primer 1). 0.5 μl and Taq polymerase (Biogrand, Seoul, Korea) were added to each of them. The reaction was amplified for 10 minutes, and the PCR product was electrophoresed on 1% LE agarose gel (FMC) and observed for amplification and size.

As the experimental results shown in Figs. 5 and 6, wherein the primers are Bacillus anthracis (B. anthracis) gene amplification and only a specific, Bacillus cereus (B. cereus) and the gene of the other Bacillus strains that do not know that amplification Could.

As described above, the detection method according to the present invention is effective in detecting only anthrax, and in particular, it is possible to distinguish B. cereus and anthrax, which are very similar to anthrax and taxonomically and molecularly. There is an advantage, it can be very useful for the detection of anthrax.

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Claims (7)

(a) a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence complementary to (a) above; And (c) at least one anthrax from the base sequence of (a) or (b) and selected from the group consisting of 20 bp or more consecutive nucleotide sequences comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44 Detection probe. The anthracnose detection probe according to claim 1, wherein the base sequence of (a) comprises SEQ ID NO: 1. Anthrax detection microarray characterized in that the probe of claim 1 or 2 is integrated on a substrate. Primer for anthrax detection having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 44. Method for detecting anthrax using at least one selected from the group consisting of the probe of claim 1, the probe of claim 2 and the primer of claim 4. The method of claim 5, wherein the detection is performed by PCR (Polymerase Chain Reaction) analysis, DNA sequencing (DNA sequencing), microarray hybridization, PCR-RFLP (restriction fragment length polymerphism) analysis, randomly amplified polymorphic DNA DNA amplification fingerprinting (DAF), arbitrarily primed PCR (AP-PCR), sequence tagged site (STS), expressed sequence tag (EST), sequence characterized amplified regions (SCAR), inter-simple sequence repeat amplication (ISSR), One selected from the group consisting of amplified fragment length polymorphism (AFLP), cleaved amplified polymorphic sequence (CAPS), single-strand conformation polymorphism (PCR-SSCP), northern hybridization and southern hybridization Method carried out by the above method. Anthrax detection composition comprising at least one selected from the group consisting of a probe of claim 1, a probe of claim 2 and a primer of claim 4.

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