CN1232651C - Clinical biochip for detecting bacteria - Google Patents

Abstract

The present invention discloses a biological chip which is characterized in that a GenBank data base is used in the design of combination probes of the chip to search ribosome nucleic acid gene sequences of 50 to 100 kinds of bacteria. Software, such as Primer Premiere, DNAStar, Clustal-X, etc., is used for designing primers and probes, comparing the sequences and conventionally synthesizing all related oligonucleotide sequences, and each kind of bacterium is provided with four probes. The synthesized probes are dissolved by 40% to 60% of DMSO, and the final concentration is 1 g/l to 3 g/l. The biological chip is arranged in a GSI Flexys sample applicator for setting a sample procedure into a point embattling type. The present invention has the advantages of specificity accuracy, high speed and sensitivity and embodies one-to-many detecting modes.

Description

A kind of biochip that is used for the clinical bacteria detection

Technical field

The present invention relates to a kind of biochip, particularly relate to a kind of biochip that clinical bacteria detects that is used for.

Background technology

Although viral infection such as acquired immune deficiency syndrome (AIDS), hepatitis and hemorrhagic fever etc. become worse, infectation of bacteria remains one of human health and threatens greatly.For example the death toll that causes of tuberculosis still occupies the No. 1 in the world, and the sign that stages a comeback is arranged; The streptococcal infection of B family is fatal often with interior newborn infant for two months; The Salmonellas of food source, listeria spp and intestinal bacteria etc. are the important factors that causes diarrhoea; The air conditioner ventilating system of large-scale totally-enclosed building is often breeding legionella pneumophilia, a kind of pathogenic bacteria of legionnaires disease; Cause that several cause of diseases of pneumonia such as Klebsiella pneumonia, pneumococcus, Chlamydia pneumoniae, Mycoplasma pneumoniae etc. often constitute very big threat to lower for the resistance, old or young crowd; Charrin disease is a modal infection type among burn, the war wound patient, and difficult with control; Hemophilus influenzae is the common bacteria in the upper respiratory tract infection, is difficult to separation and Culture; Infection of staphylococcus aureus is plain stubborn, and resistance is very general; Meningococcus threatens bigger to the still very imperfect children of resistibility.The diagnosis of bacterial infection and detection have important directive significance for clinical treatment.Although various Broad spectrum antibiotics is arranged, so-called wide spectrum also has certain sphere of action, is not all effective to all clinical bacterias; And microbiotic abuse problem is more and more outstanding, uses the New-type wide-spectrum microbiotic to hide some dangers for for later antibiotic therapy simply, and when reusing such microbiotic, its effect just can not normally be brought into play again.

In case having established is the infection of any bacterium, just can take out treatment plan with strong points, infectation of bacteria will soon be effectively controlled.Therefore the detection of infectious bacteria kind has crucial clinical guidance meaning.The detection method of bacterium is varied, the primary goal of qualitative detection yes Clinical Laboratory; Detection by quantitative has more deep understanding for number of bacteria and gradient of infection.The method that has not only can be qualitative but also can be quantitative, as enzyme linked immunosorbent assay (ELISA, enzyme-linkedimmunosorbent assay), polymerase chain reaction (PCR, polymerase chain reaction) and nucleic acid hybridization etc.Every kind of Bacteria Detection means all have the relative merits of oneself, can not rely on single means to come bacterium is carried out qualitative, and any detection method all is a factor in the numerous methods of clinical conclusive evidence.There are two in classical way than obvious defects: the one, and detection time is generally longer, and the 2nd, belonging to man-to-man detecting pattern is that single test can only detect a kind of bacterium.

Summary of the invention

The object of the invention is to provide a kind of oligonucleotide combination probe micro-array biochip, can be used for the disposable detection of multiple pathogenetic bacteria, improves the accuracy and the reliability that detect, shortens the classical required time of detecting.

The present invention seeks to be achieved through the following technical solutions:

Biochip involved in the present invention be with bacterial 16 S ribosomal nucleic acid serve as the amplification target design.16S rrna nucleic acid extensively is present in these class prokaryotic organism of bacterium, for example all has the 16S ribosomal rna sequence in archeobacteria, eubacterium, rickettsia, mycoplasma, chlamydozoan, spirochete and the actinomycetes.Ribosomal rna sequence is alternately arranged by conserved sequence and variable sequence and is constituted, and we are located in the variable region by designed oligonucleotide combination probe.Selected rrna nucleic acid variable region variation specific sequence designing probe greatly, at 4 of every kind of bacterium designs, 4 characteristic variable regions of corresponding bacterial ribosome nucleic acid respectively.Use corresponding information biology software design and go out the probe of multiple pathogenetic bacteria, then probe and GenBank database are compared, tentatively determine the probe feasibility according to return message.Transferring to commercialization company after probe is determined, to carry out nucleotide sequence synthetic.Synthetic good probe uses GSI Flexys high-density point sample instrument to carry out point sample.

Concrete steps are as follows:

The design of 1 combination probe: utilize the GenBank database, the rrna nucleic acid gene sequence of retrieval various bacteria, number of bacteria can be decided according to the practical application needs, for example the 50-100 kind.Use Primer Premiere, DNAStar, softwares such as Clustal-X carry out primer, probe design and sequence relatively, conventional synthetic all oligonucleotide sequences that relate to.4 probes of every kind of bacterium design.

Serve as the amplification target with 16S rRNA in the combination probe of the present invention design, the universal amplification primer is S1:5 '-AGA GTT TGA TC (A/C) TGG CTC AG-3 ' and S2:5 '-CCG TCA ATT C (A/C) T TT (A/G) AGT TT-3 ', and the amplified fragments size is 890-bp-930-bp.

2 point samples: will synthesize good probe and dissolve with 40-60%DMSO, ultimate density is 1-3g/l.Biochip is placed GSI Flexys point sample instrument, the point sample program is set, the point sample of structuring the formation is as accompanying drawing point sample form.Make gene chip of the present invention.

The using method of gene chip of the present invention is, various clinical samples are comprised that blood, cerebrospinal fluid, ascites, phlegm, collutory, chest hydrops etc. extract nucleic acid after treatment, adopt the method for polymerase chain reaction (PCR) that fluorescein (Cy5-dCTP) is incorporated in the PCR product, the PCR product is added to through thermally denature and carries out hybridization on the biochip.Reacted chip uses chip scanner scanning, judges the kind of pathogenetic bacteria according to results of hybridization.

Biochip of the present invention utilizes nucleic acid amplification in vitro technology, and the specificity of bind nucleic acid hybridization can be finished detection time in 6 hours; And can easily hold hundreds and thousands of specific probes, single test can detect a plurality of targets, need not repetition, and therefore required sample size also has only portion.Use combination probe to improve the specificity that detects.Differentiate meaning although bacterial ribosome nucleic acid has certain kind, because its conservative property, its rrna nucleic acid of kind in very near source was quite similar on some was evolved.It is very difficult to adopt a probe to distinguish these nearly derived bacteriums separately, relies on the combination of 4 probes to come corresponding a kind of bacterium, has improved the accuracy that detects greatly.This has crucial meaning for overcoming cross reaction and the false positive results that ordinary method hybridization produced.

The related bacterium of the embodiment of the invention all is significant clinically pathogenic bacterium.The conventional sense means waste time and energy, and order of accuarcy is not high, and biochip of the present invention need not repetition and trial, thereby tool has an enormous advantage in Clinical Laboratory because primary first-order equation just can detect related bacterium.In addition, have the cause of disease that causes important transmissible disease, be the plague that rises year by year, the enteron aisle prevailing disease that generation is all arranged every year, bacterial food poisoning etc. as anthrax bacillus and Brucella, the outbreak of epidemic danger that causes the infecting both domestic animals and human disease in the pastoral area in China.For the diagnosis of these strong pathogenic agent, domestic also do not have simple and rapid diagnostic reagent commodity.Biochip of the present invention can be used for multinomial fields such as customs quarantine control, health and epidemic prevention, environmental monitoring, food inspection equally, thereby has huge market outlook.

The terrorist utilizes strong pathogenic agent to cause great threat for country and social safety as the bioterrorism event of bio-terrorism means manufacturing, and Biosafety and bio-terrorism defend to have become national governments and the country and the social safety major issue of numerous people common concern today.The strong pathogenic agent that rapid detection goes out in the bioterrorism event is most important to the harm of taking correct means elimination terrorist incident to cause, biochip of the present invention will help to improve the ability that we tackle the burst biological event, can calm down people's psychology fear, prevent to cause social unrest, guarantee social safety that the social agency of its generation is very huge with influence and effectively prevent and control bioterrorism event.Except that bioterrorism event, the eruption and prevalence of deadly infectious disease, tend to make a lot of people's infection even seize a lot of people's life, also can cause people huge psychological fear and social unrest, make diagnosis fast, the control disease of taking measures is popular can produce powerful social agency and influence equally.

Advantage of the present invention is embodied in:

Special accurate: as because the present invention has adopted combination probe, to point to a bacterium, therefore increased detection specificity greatly by many probes.

Fast: biochip test of the present invention comprises sample pre-treatments, pcr amplification and chip hybridization, about 6 hours consuming time altogether, conventional bacterium separation and Culture needs about 3 days, and clinical bacteria to be separated into power lower, some bacterium separation and Culture that What is more needs two weeks to month, as mycobacterium tuberculosis.The present invention can shorten Diagnostic Time.

Sensitive: biochip of the present invention combines the pcr amplification technology, therefore has higher sensitivity.

The one-to-many detecting pattern: all bacteriums in the single test testing goal sample, reduce sample size, reduced simultaneously because the expensive and time of repeatedly attempting bringing.Though nonrecurring cost is high relatively slightly, if consider detection when being tens kinds of bacteriums, its relative cost remains low-down.

Following experimental example is used to further specify the present invention.

Experimental example 1:The hybridization of pure cultures of bacteria

Cultivate 80 kinds of bacteriums according to pertinent literature.Use QIAGEN DNeasy Tissue Kit (Cat.No.69504) to extract bacterial chromosomal dna.Use the corresponding bacterial chromosomal dna of S1,10mM Tris-HCl (pH 8.4) with the S2 primer amplification; 50mM KCl; 1.5mM MgCl ₂Gelatin 0.01%; TritonX-100 0.1%; Tween 20 0.01%; 40 μ M of dATP, dTTP and dGTP, 30 μ M ofdCTP (Promega), 10 μ M of Cy5-dCTP (Amersham Pharmacia Biotech), 0.1 μ M of each primer, 2U of Taq polymerase (worker's biotechnology company limited is given birth in Shanghai) and 15ng amplification template.The PCR parameter is 95 ℃ of pre-sex change, and 15min is 94 ℃ then, 10s; 63 ℃, 10s; 72 ℃, 15s; 29 circulations.Product uses Amicon Microcon-PCR colums, and (final elution volume is 25 μ l for Millipore, USA) purifying.Getting amplified production 10 μ l behind fluorescent mark and the purifying joins and forms the hybridization working fluid in the 90 μ l hybridization solutions, chip is put into GSI GeneTACHybridization Station hybridization instrument hybridizes, at 80 ℃ of adding probes, pre-sex change 10min, hybridization temperature is 45 ℃, hybridization time 1 hour.Take out biochip after finishing, use the scanning of GSI GeneTAC1000 biochip scanner, analysis and saving result.Utilize the results of hybridization of pure cultures of bacteria to show that biochip of the present invention can relate to major part Bacteria Identification to the position of planting.

Experimental example 2:Sensitivity test

With LB inclined-plane switching streptococcus aureus and Pseudomonas aeruginosa, cultivated 12 hours for 37 ℃.Scrape with transfering loop and to get one and encircle among the 5ml 0.03mol/L PBS, do 10 times of serial dilutions then until 10-9.Utilize dull and stereotyped streak method that two kinds of bacteriums are carried out enumeration.Get each extent of dilution and carry out the chip hybridization experiment, chip hybridization sensitivity behind the observation bacterium serial dilution.Get legionella pneumophilia chromosomal DNA 15ng/ml, 1.5ng/ml, 150pg/ml, 15pg/ml, 1.5pg/ml it is fluorescein-labelled that four extent of dilution carry out pcr amplification and Cy5 respectively, uses fluorescein-labelled thing and chip hybridization then, observes the hybridization sensitivity of karyomit(e) dilution back.Through plate count, calculate bacterium liquid original concentration.Streptococcus aureus is 2.35 * 10 ⁷CFU/ml (colony forming unit), Pseudomonas aeruginosa is 2.0 * 10 ⁷CFU/ml.For two kinds of bacterium liquid, biochip test sensitivity is 235 and 200CFU.And simulation blood specimen sensitivity, 150pg/ml karyomit(e) can be observed positive signal through PCR, though and 15pg/ml PCR has not observed band, biochip still can detect positive signal, highly sensitive 10 times than PCR-electrophoretic method of biochips.

Experimental example 3:The specificity test

Use Mycobacterium bovis, Mycobacterium intracellulare, mycobacterium avium and these several bacterial classifications close with mycobacterium tuberculosis of mycobacterium littorale to carry out chip hybridization, observation and mycobacterium tuberculosis intersect situation.Test-results shows, hybridizes with in the Mycobacterium bovis of the nearly edge of mycobacterium tuberculosis and Mycobacterium intracellulare and 4 probes of mycobacterium tuberculosis 3, and mycobacterium littorale and mycobacterium avium are only hybridized with 2 probes.That is to say that combination probe has played critical discriminating effect here, if do not use combination probe, the close bacterium of the so multiple class of very difficult differentiation.

Experimental example 4:Practical application

Biochip of the present invention has passed through simulation blood specimen and double-blind method test, proves that it has sizable reliability.The streptococcus aureus and the Pseudomonas aeruginosa bacterium liquid 20 μ l of serial dilution in the experimental example 2 are joined in the 180 μ l horse blood, form the simulation blood specimen of series concentration.Extract the test kit operation instructions according to the QIAamp whole blood DNA and extract DNA, and it is fluorescein-labelled to carry out Cy5 as template, carries out chip hybridization according to the method described above, observes simulation blood specimen hybridization sensitivity.Simulation blood specimen sensitivity is respectively 23500 and 2000CFU.

Get the chromosomal DNA of 3 unknown samples, label is 1,2 and 3.Carry out 1,2,3,1+2,1+3,2+3 and 1+2+3 combination, wherein built-up sequence and mode the unknown finished sample combination by an other people.This 8 duplicate samples (moisture) is simulated Blind Test, with the array mode contrast, judges the feasibility that various product detect then.Compound sample Blind Test experimental result shows, if exist more than one object to be measured also can detect simultaneously in the sample.Two kinds of bacterium mixing energies detect simultaneously, and three kinds of bacterium mix and generally can detect wherein dominant two kinds of bacterium simultaneously.If several bacterial concentrations in actual the infection are roughly consistent, then biochip can detect them simultaneously.

Experimental example 5:

Two parts of certain hospital's censorship samples, suspecting has Pseudomonas aeruginosa.Through after the biochip hybridization, find that 4 probes of a strain bacterium and Pseudomonas aeruginosa are hybridized, another strain bacterium then hybridizes with 4 probes of salmonella typhi.Thereby be judged to be Pseudomonas aeruginosa and salmonella typhi respectively.Find after the microbial culture that the former produces green pigment, and the latter does not have, this is consistent with the feature of Pseudomonas aeruginosa.Judgement with classical Bacteria Identification program and nucleic acid authentication method proof and biochip matches then.

Description of drawings:

Accompanying drawing 1 is a combined probe type biochip Pareto diagram, wherein the positive control point of point in the solid box.

Embodiment

Embodiment:

1, probe design: utilize the GenBank database, the rrna nucleic acid gene sequence of 80 kinds of bacteriums of retrieval, use Primer Premiere, DNAStar, softwares such as Clustal-X carry out primer, probe design and sequence relatively, and it is synthetic by the living worker's biotechnology in Shanghai company limited that all relate to the synthetic oligonucleotide sequence.16S rRNA universal amplification primer is S1:5 '-AGA GTT TGA TC (A/C) TGGCTC AG-3 ' and S2:5 '-CCG TCA ATT C (A/C) T TT (A/G) AGT TT-3 ', and the amplified fragments size is 890-bp-930-bp.80 kinds of bacteriums are designed 320 probes (seeing attached list 2) altogether.4 probes of every kind of bacterium design.

Point sample: will synthesize good probe and dissolve with an amount of 50%DMSO, ultimate density is 2g/l.Biochip places GSI Flexys point sample instrument, and the point sample program is set, and the point sample of structuring the formation is put to become as accompanying drawing sample form.Wherein the point sample corresponding relation of 80 kinds of bacteriums is as shown in table 1::

Table 1: built-up type oligonucleotide probe and accompanying drawing corresponding relation

	The dog brucella	The hot rickettsia of Q	The pneumophila legionella pneumophilia	The fluorescens Pseudomonas fluorescens	The aureus streptococcus aureus
						I	Brucella melitenis alcaligenes melitensis	Ehrlichia canis dog Ai Like body	Listeria monocytogenes monocyte hyperplasia listeria spp	Pseudomonas putida pseudomonas putida	Staphylococcus epidemidis staphylococcus epidermidis
J	Burkholderia cepacia onion bulkholderia cepasea	Enterobacter aerogenes enteroaerogen	Moraxella catarrhis morazella catarrhalis	Pseudomonas stutzeri Pseudomonas stutzeri	Stenotrophomonas maltophilia has a liking for the few food of Fructus Hordei Germinatus Zymomonas mobilis
						K	Burkholderia pseumonallei pseudoglanders bulkholderia cepasea	Enterobacter cloacae enterobacter cloacae	Mycobacterium avium mycobacterium avium	Rickettsia prowazekii Rickettsia prowazeki	Streptococcus agalactiae streptococcus agalactiae
L	Chlamydia pneumoniae Chlamydia pneumoniae	Enterococcus faecalis enterococcus faecalis	Mycobacterium bovis Mycobacterium bovis	The vertical rickettsia of Rickettsia rickettsia	Streptococcus pneumoniae streptococcus pneumoniae
						M	Chlamydia psittaci chlamydia psittaci	Enterococcus faecium faecium	Mycobacterium intracellular Mycobacterium intracellulare	Salmonella paratyphi salmonella paratyphi	Streptococcus pyogenes streptococcus pyogenes
N	Chlamydia trachomatis sand holes chlamydozoan	Escherichia coli intestinal bacteria	Mycobacterium smegmatis M. smegmatics	Salmonella typhi salmonella typhi	Streptococcus salivarium streptococcus-salivarius
						O	Citrobacter amalonaticus does not have the propanedioic acid citric acid bacillus	The graceful pseudomonas fluorescens of the rich taste of Fluoribacter bozemanae	Mycobacterium tuberculosis mycobacterium tuberculosis	Salmonella typhimurium Salmonella typhimurium	Yersinia enterocolitica yersinia entero-colitica
P	Citrobacter freundii citrobacter freundii	Francisella tularensis soil draws not Salmonella	Mycobacterium xenopi mycobacterium littorale	Serratia liquefaciens liquefied Serratia	Yersinia pestis Yersinia pestis

Annotate: A1 to A4 represents 1 to No. 4 probe of E.coli, and A5 to A8 represents 1 to No. 4 probe of Citrobacter koseri, and the rest may be inferred by analogy.

Subordinate list 2:80 kind bacterial 16 S rRNA specific probe

Bacterial species	1	2	3	4
					1.Acinetobacter calcoaceticus acinetobacter calcoaceticus	CGGGGGAAGGTAGCTT GCTACCG	CCTAGAGATAGTGGAC GTTAC	GGGGAAGGGCAGCCAT CTGGC	GGGCCTTTGAGGCTTTAG TGGCGC
2.Acinetobacter haemolyticus acinetobacter haemolyticus	GTTGGGGCCTTTGAGG CTTTAGTG	CTACTTTAGTTAATAC CTAGAGATA	TCCTACGGGAGAAAGC AGGGGATC	AGAGAGGTAGCTTGCTAC TGATCT
					3.Bacillus anthracis Bacillus anthracis	AAGTGTTAGAGGGTTT CCGCCCT	AAGTGCTAGTTGAATA AGCTGGCAC	AATTGAAAGGCGGCTT CGGCTGT	GCGAATGGATTAAGAGCT TGCTCT
4.Bacteroides fragilis bacteroides fragilis	TTTGCGATATACAGTA AGCGGCCA	ATAAAGTGCAGTATGT ATACTGTTT	TAATGATTCCGCATGG TTTCATTA	AAGCTTGCTTTCTTTGCT GGCGA
					5.Bartonella henselae Han Saiba entire body	GGCGGTTTACTGCTCG GTGGCGCA	ACCGGTGAAGATAATG ACGGTAACC	CGTATACGTCCTATTT GGAGAAAG	GCGCACTCTTTTAGAGTG AGCG
6.Bartonella quintana trench fever crust entire body	GGGTGGTTTACTACTC GGTGGCG	Same B.henselae	TATACGTCCCTCTGGG AGAAAGA	Same B.henselae
					7.Brucella abortus Bacillus abortus	CCGTCGGGGTGTTTAC ACTTCGG	GGAGAAGATAATGACG GTAACCCGA	ATGTGCTTGGGGGAAA GATTTATC	CGAGCGCCCGCAAGGGTG AGCG
8.Brucella canis dog brucella	B.abortus	GGTGAAGATAATGACG GTAACCGGA	GTGCCCTTCGGGGGAA AGATTTAT	CGAGCGCCCCGCAAGGGG AGC
					9.Brucella melitenis alcaligenes melitensis	B.abortus	Same B.canis	Same B.canis	Same B.canis
10.Burkholderia cepacia onion bulkholderia cepasea	TTGTTGGGGATTCATT TCCTTAG	AAATCCTTGGCTCTAA TACAGTCG	CATACGATCTACGGAT GAAAGCG	CACGGGTGCTTGCACCTG GTGG
					11.Burkholderia	Same B.cepacia	AATCATTCTGGCTAAT	ATACGATCTGAGGATG	GCACGGGCTTCGGCCTGG

Pseumonallei pseudoglanders bulkholderia cepasea		ACCCGGAGT	AAAGCG	TG
					12.Chlamydia pneumoniae Chlamydia pneumoniae	TTGATGTGGATGGTCT CAACCCCA	AAGAGAGATTGGCTAA TATCCAAT	TAATTAGGCATCTAAT ATATATTA	GAATAATGACTTCGGTTG TTATTT
13.Chlamydia psittaci chlamydia psittaci	CATACTTGATGTGGAT AGTCTCAA	Same C.pneumoniae	TTTAGGCATCTAAAAC ATATTAAA	Same C.pneumoniae
					14.Chlamydia trachomatis sand holes chlamydozoan	Same C.pneumoniae	GAAGACGGTTAATACC CGCTGGATT	ATATTTGGGCATCCGA GTAACGTT	AGCAATTGTTTCGACGAT TGTTT
15.Citrobacter amalonaticus does not have the propanedioic acid citric acid bacillus	ATGTCTATTTGGAGGT TGTGCCCT	AAGGGGTTAAGGTTAA TAACCTTAG	ATAATGTCGCAAGACC AAAGAGGG	TAACAGGAAGCAGCTTGC TGCTTT
					16.Citrobacter freundii citrobacter freundii	CGATGTCGACTTGGAG GTTGTGC	AAGGCGTTGTGGTTAA TAACCGCAG	Same C.amalonaticus	TAGCACAGAGGAGCTTGC TCCTTG
17.Citrobacter the suitable citric acid bacillus of koseri	Same C.freundii	AAGGTGTTGTGGTTAA TAACCACAG	Same C.amalonaticus	Same C.amalonaticus
					18.Clostridium botulinum Clostridium botulinum	TAGGGGGTATCAACTC CCCCTGT	The same	ATTTTCTTATCAAAGA TTTATTGCTT	The same
19.Clostridium perfringes clostridium perfringens	GGGGTTTCAACACCTC CGTGCCG	TCTGTCTTTGGGGAAG ATAATGACG	CATCATCATTCAACCA AAGGAGCA	ATGAAGTTTCCTTCGGGA AACG
					20.Clostridium tetani clostridium tetani	GGTGTAGGAGGGTCCA ACCTTCT	TTTCTGGGACGATAAT GACGGTAC	ACTTTAACCAAAGGAG TAATCTGCTT	CGAGCGATGAAGCTTCCT TCGGGA
21.Corynebacterium	TGAGGGTCTTCCACGA	AAGCTTTTGTGACGGT	TGTGGTGGAAAGTTTT	AAAGGCCTAGCTTGCTAG

The diphtheriae diphtheria corynebacterium	CTTTCGTG	ACCTAGATA	TCGGTACG	GTACT
					22.Corynebacterium pseudodiphtheriae corynebacterium diphtheroides	TAGGGACCTTTTGGGG TTTCTGTG	AAGCGTTTTGTGACGG TACCTGGAG	GTGGAAAGTTTTTTCG GTGTAGGA	GAAAGGCCTTCGGAGG TACTC
23.Corynebacterium xerosis Corynebacterium xerosis	TAGGGGTCTTCCACGA CTTCTGT	CATCGACGAAGGTTTT CTGACGGT	CGCACCGTGAGGGTGT GGTGGAAA	CCCCAGCTTCCTGGGGTA CACGA
					24.Coxiella the hot rickettsia of burnetii Q	TTGGGAAGTTCACTTC TTAGTAGC	TTCTCAAGGGTAATAT CCTTGGGCG	CATAATCTCTTTGGAG CAAAGCG	GGAGCTTGCTCCTGGCGG CGAG
25.Ehrlichia canis dog Ai Like body	TGAGGATTTTATCTTT GTATTGTA	TAATGACGGTACCTAT AGAAGAAGT	ACTGTATAATCCCCGA GGGGGAA	CCTCTGGCTATAGGAAAT TGTT
					26.Enterobacter aerogenes enteroaerogen	Same K.pneumoniae	GGCGTTAAGGTTAATA ACCTTGGCG	TAACGTCGCAAGACCA AAGTGGG	CACAGAGAGCTTGCTCTC GGGT
27.Enterobacter cloacae enterobacter cloacae	Same K.pneumoniae	GGTGTTGTGGTTAATA ACCGCAGCA	Same E.aerogenes	Same E.aerogenes
					28.Enterococcus faecalis enterococcus faecalis	TGGAGGGTTTCCGCCC TTCAGTGC	GGACGTTAGTAACTGA ACGTCCCCT	AGTTTATGCCGCATGG CATAAGAG	TTCCTCCCGAGTGCTTGC ACTC
29.Enterococcus faecium faecium	E.faecalis	ATGAGAGTAACTGTTC ATCCCTTG	TCGAAACCGCATGGTT TTGATTTG	TTTTCCACCGGAGCTTGC TCCA
					30.Escherichia coli intestinal bacteria	Same K.pneumoniae	GAGTAAAGTTAATACC TTTGCTCAT	TCTTGCCATCGGATGT GCCCA	GAAGAAGCTTGCTCTTTG CTGA
31.Fluoribacter the graceful pseudomonas fluorescens of the rich taste of bozemanae	TTGGTGATGTGAAAAT CATTAG	CAGTGGGGAGGAGGTT YGCTAGGT	ATGTCTAAGGACGAAA GCTGGGGA	AAGTCGAACGGCAGCATG ACCT
					32.Francisella tularensis soil draws	GAGTCGGTGTAAAGGC TCTAGTGG	GCCTCAAGGTTAATAG CCTTGGGGA	ATCTGTGGATTAAAGG TGGCTTTC	TAACAGGTCTTAGGATGC TGAC

Salmonella not
					33.Haemophilus influenzae hemophilus influenzae	GGATTGGGCTTAGAGC T	TTGATGTGTTAATAGT ACATCAAAT	AGATGAAAGTGCGGGA CTGAGAGG	AGCAGGAGAAAGCTTGCT TTCTT
34.Haemophilus parainfluenzae haemophilus parainfluenzae	CTTTTAGCTTGGCGCC CGTAGC	GCATTTAGTTTAATAG ACTAGGTGAT	TGATCGGGAGATGAAA GTGTGGG	AACATGAAGAAGCTTGCT TCTTT
					35.Hafnia alvei honeycomb Ha Funiya bacterium	Same K.pneumoniae	AGGGAGTAAAGTTAAT ACCTTTGC	CCTTAGGGCCTCTTGC CATCGGAT	TAACAGGAAACAGCTTGC TGTTT
36.Helicobacter pylori helicobacter pylori	GAGGGCTTAGTCTCTC CAGTAATG	TTGTTAGAGAAGATAA TGACGGTAT	GATACTCCTACGGGGG AAAGATTT	ATGAAGCTTCTAGCTTGC TAGA
					37.Klebsiella oxytoca produces sour Cray uncle formula bacterium	GTTGTTCCCTTGAGGA GTGGCTT	CATTAAGGTTAATAAC CTTAGTG	GCATAACGTCGCAAGA CCAAAGA	TAGCACAGAGAGCTTGCT CTCGG
38.Klebsiella pneumoniae kerekou pneumonia uncle formula bacterium	GTTGTGCCCTTGAGGC GTGGCT	AAGGCGATGAGGTTAA TAACCTCATC	CAAAGTGGGGGACCTT CGGGCCTC	Same K.oxytoca
					Cold gram Lu Wal Salmonella 39.Kluyvera cryocrescens is dwelt	Same Kpneumoniae	GGCATTAAGGTTAATA ACCTTAGT	Same Eaerogenes	TAGCACAGAGAGCTTGCT CTTGG
40.Legionela pneumophila legionella pneumophilia	TGGTTATATGAAAATA ATTAGT	AGGGTTGATAGGTTAA GAGCTGATTAA	TGTCTGAGGACGAAAG CTGGGGAC	ATGCAAGTCGAACGGCAG CAT
					41.Listeria monocytogenes monocyte hyperplasia listeria spp	TAGGGGGTTTCCGCCC CTTAGTGC	ATAAGAGTAACTGCTT GTCCCTTGAC	TGTGGCGCATGCCACG CTTTTGAA	ACGGAGGAAGAGCTTGCT CTTCC
42.Moraxella catarrhis morazella catarrhalis	AGTTAGGAAGCTTGCT TCTGATA	ACCCATAAGCCCTGAC GTTACCCA	ATGGCGAAGGCAGCTC CCTGGC	TGGGTCTTTTAAAGACTT AGTGAC

43.Mycobacterium avium mycobacterium avium	Same Mtuberculosis	Same Mtuberculosis	CATGTCTTCTGGTGGA AAGCTTTT	AAGGCCTCTTCGGAGGTA CTC
					44.Mycobacterium bovis Mycobacterium bovis	Same Mtuberculosis	Same Mtuberculosis	TGTCTTGTGGTGGAAA GCGCTTTA	AAAGGTCTCTTCGGAGAT ACT
45.Mycobacterium intracellula Mycobacterium intracellulare	Same Mtuberculosis	Same Mtuberculosis	GGACCTTTAGGCGCAT GTCTTTAG	GAAAGGCCCCTTCGGGGG TACT
					46.Mycobacterium smegmatis M. smegmatics	Same Mtuberculosis	AGCACAGACGAAGCGC AAGTGA	CACCCTGCTGGTCGCA TGGCCTGG	AAAGGCCCTTTCGGGGGT ACT
47.Mycobacterium tuberculosis mycobacterium tuberculosis	TAGGTGTGGGTTTCCT TCC	GGTCCGGGTTCTCTCG GATT	GATAGGACCACGGGAT GCATGTCT	Same Mbovis
					48.Mycobacterium xenopi mycobacterium littorale	GTTCTTTCCTGAAGGA TCCGTGCC	TTCAGCCTCGACGAAG CTGCG	TAGGACCATTCTGCGC ATGTGGG	AGGCCCCTTTTTTGGGGT GCT
49.Mycoplasma pneumoniae Mycoplasma pneumoniae	GTCGGGGCGATCCCCT CGGTAGTG	AATGACTTTAGCAGGT AATGGCTA	TTGGTTCGCATGAATC AAAGTT	TCGAAAGTAGTAATACTT TA
					50.Neisseria gonorrhoeae gonococcus	GTCAATTAGCTGTTGG GCAACT	AGGCCGTTGCCAATAT CGGCGGCCGAT	ACGTCTTGAGAGAGAA AGCAGGGG	CACAGGGAAGCTTGCTTC TCG
51.Neisseria meningitidis Neisseria meningitidis	GGCAACTTGATTGCTT AGTA	GGCTGTTGCTAATATC AGCGGCTGATG	ATACGTCTTGAGAGAG AAAGCAGG	GCACAGAGAAGCTTGCTT CTC
					52.Orientia tsutsugamushi tsutsugamushi fever east body	TGGGGGATTTTTCTTT CAGTT	TAGTAGGGATGATAAT GACAGTACC	TATGCCCTCTATAAGG AGGAAAG	AATGCTGAGTTTGCTTAG TATT
53.Pasteurella multocida multocida	ATTGGGCTATATGCTT GGTGCCCG	GATGTTGTTAAATAGA TGGCATCAT	ATCTCTGAGGAGTAAA GGGTGG	TAGCAGGAAGAAAGCTTG CTTTC

54.Proteus mirabilis proteus mirabilis	ACAGGAGAAAGCTTGC TTTCTTG	ACCCTTATCAATTGAC GTTACC	CCGGTGGCGAAGGCGG CCCCC	TTGTGGTCTTGAACCGTG GCTTCT
					55.Pseudomonas aeruginosa Pseudomonas aeruginosa	CCGTTGGGATCCTTGA GATC	GGGCAGTAAGTTAATA CCTTGCTGT	CATACGTCCTGAGGGA GAAAGTG	ATGAAGGGAGCTTGCTCC TGGAT
56.Pseudomonas fluorescens Pseudomonas fluorescens	CCGTTGGGAGCCTTGA GCTCTT	CATTAACCTAATACGT TAGTGTT	TACGTCCTACGGGAGA AAGCAG	AGAGAGAAGCTTGCTTCT CTTGA
					57.Pseudomonas putida pseudomonas putida	GCCGTTGGAATCCTTG AGATTT	Same Pfluorescens	Same Pfluorescens	GAGAAGAGCTTGCTCTTC GATTC
58.Pseudomonas stutzeri Pseudomonas stutzeri	Same Paeruginosa	Same Paeruginosa	TCCTACGGGAGAAAGT GGGGGA	TGAGTGGAGCTTGCTCCA TGATT
					59.Rickettsia prowazekii Rickettsia prowazeki	ATCGGAGGATTCTCTT TCGGTTTC	ATAATGACGTTACTTG CAGAAAAAG	TATTCTCTACGGAGGA AAGATT	AACTAGAGCTCGCTTTAG TTAA
60.Rickettsia the vertical rickettsia of rickettsia	Same Rprowazekii	Same Rprowazekii	TATATTCTCTGCGGAG GAA	AATTGGGGCTTGCTCCAA TTAG
					61.Salmonella paratyphi salmonella paratyphi	GGTTGTGCCCTTGAGG CGTGGC	Same Styphi	TCGCAAGACCAAAGAG GGGGACTT	Same Styphimurim
62.Salmonella typhi salmonella typhi	Same Sparatyphi	TGTTGTGGTTAATAAC CGCAGCAAT	Same Styphimurium	GAAGCAGCTTGCTGCTTT GCTGA
					63.Salmonella typhimurium Salmonella typhimurium	Same Sparatyphi	Same Styphi	AAGACCAAAGAGGGGG ACCTTCGG	GAAGCAGCTTGCTCTTTG CTGA
64.Serratia liquefaciens liquid	Same Kpneumoniae	AGGGTTCAGTGTTAAT AGCACTGTT	ATAACGTCTACGGACC AAAGTGGG	GAAGCTTGCTCTCTGGGT GACGA

Change Serratia
					65.Serratia marcescens serratia marcescens	Same Kpneumoniae	GGTGGTGAGCTTAATA CGTTCATCA	Same Eaerogenes	GCACAGGGGAGCTTGCTC CCTGG
66.Serratia plymuthica Plymouth Serratia	Same Kpneumoniae	GAAGGGCAGTGTGTTA ATAGCACAT	Same Sliquefaciens	AGCACAGGAGAGCTTGCT CTCTG
					67.Serratia rubidaea serratia rubida	Same Kpneumoniae	AGGAGGAAGGTGGTGA ACTTAATACG	Same Eaerogenes	GGAGGAAGCTTGCTTCCT CGCCG
68.Shigella boydii Shigella bogdii	Same Kpneumoniae	AGGGAGTAAAGTTAAT ACCTTTGCTC	GAGGGGGACCTTCGGG CCTCTTG	CAGGAAGCAGCTTGCTGT TTCGC
					69.Shigella dysenteriae shigella dysenteriae	Same Kpneumoniae	Same Sboydii	Same Sboydii	CAGAAAGCAGCTTGCTGT TTGC
70.Shigella flexneri shigella flexneri	Same Kpneumoniae	Same Sboydii	Same Sboydii	CAGGAAGCAGCTTGCTGT TTCG
					71.Shigella Shigellae in Song sonnei	Same Kpneumoniae	Same Sboydii	Same Sboydii	CAGGAAACAGCTTGCTGT TTCG
72.Staphylococcus aureus streptococcus aureus	TTAGGGGGTTTCCGCC CCTTAGT	TAAGTAACTGTGCACA TCTTGACG	TATTTTGAACCGCATG GTTCAAAA	GGACGAGAAGCTTGCTTC TCTGAT
					73.Staphylococcus epidemidis staphylococcus epidermidis	Same Saureus	AATGTGTAAGTAACTA TGCACGTC	TATATTGAACCGCATG GTTCAATA	AGACGAGGAGCTTGCTCC TCTGAC
74.Stenotrophomonas maltophilia has a liking for the few food of Fructus Hordei Germinatus Zymomonas mobilis	ACAGTAAGAGCTTGCT CTTATG	CTCGGTTGTTCTGACG GTACCC	CGAAGGCAGCTACCTG GACCAAC	GTTGGGTGCAATTTGGCA CGCAGT
					75.Streptococcus agalactiae agalasisa	TTAGGCCCTTTCCGGG CGTTAGT	TAGGAGTGGAAAATCT ACCAAGTG	AAGAGTAATTAACACA TGTTAGTT	CACCTAGATCCTGATGAG TT

Suis
					76.Streptococcus pneumoniae streptococcus pneumoniae	GGTGTTAGACCCTTTC CGGGGTC	TGTGAGAGTGGAAAGT TCACACTG	AAGAGTAGATGTTGCA TGACATTT	AAGTAGAACGCTGAAGGA AGGAGC
77.Streptococcus pyogenes streptococcus pyogenes	TGTTAGGCCCTTTCCG GGGCTTA	GTGGGAGTGGAAAATC CACCAAGT	TAAGAGAGACTAACGC ATGTTAGT	AACTGGTGCTTGCACCGG TTCAAG
					78.Streptococcus salivarium streptococcus-salivarius	GTTGGATCCTTTCCGG GATTCAG	Same Spneumoniae	AATGGATGACCCATGT CATTTATT	GAGAGGAGCTTGCTCTTC TTGGAT
79.Yersinia enterocolitica yersinia entero-colitica	Same Kpneumoniae	AAGGCCAATAACTTAA TACGTTGT	CATAACGTCTTCGGAC CAAAGTG	AAGTAGTTTACTACTTTG CCGGC
					80.Yersinia pestis Yersinia pestis	Same Kpneumoniae	AAGGGGTTGAGTTTAA TACGCTCAA	CATGACCTCGCAAGAG CAAAGTG	GGAAGTAGTTTACTACTT TGCC

Claims (2)

1, a kind of biochip is characterized in that this chip is made by following steps:

Utilize the GenBank database, the rrna nucleic acid gene sequence of 80 kinds of bacteriums of retrieval uses Primer Premiere, DNAStar or Clustal-X software to carry out primer, probe design and sequence relatively, synthetic all oligonucleotide sequences that relate to; 16S rRNA universal amplification primer is S1:5 '-AGA GTT TGA TC (A/C) TGG CTC AG-3 ' and S2:5 '-CCG TCA ATT C (A/C) T TT (A/G) AGT TT-3 ', and the amplified fragments size is 890-bp-930-bp; 80 kinds of bacteriums amount to 320 probes, are made up of specification sheets subordinate list 2 described gene orders respectively;

Point sample: will synthesize good probe and dissolve with an amount of 50%DMSO, ultimate density is 2g/l; Biochip is placed GSI Flexys point sample instrument, the point sample program is set, put into the cloth configuration.

2, any biochip as claimed in claim 1 is characterized in that this biochip is used for Bacteria Detection.