CN101560557A - Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof - Google Patents
Genetic liquid phase chip for joint detection of five drastic pathogenic bacteria and detection method thereof Download PDFInfo
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Abstract
The invention discloses a genetic liquid phase chip for rapid detection of five drastic pathogenic bacteria of bacillus anthracis, yersinia pestis, brucella bacteria, francisella tularensis and burkholderia pseudomallei. The liquid phase chip for detecting the five drastic pathogenic bacteria has the advantages of large flux, less required sample capacity, strong specificity, high sensitivity, accuracy, high efficiency, and the like.
Description
Technical field
The present invention relates to the method for the gene liquid chip of a kind of joint-detection Bacillus anthracis, yersinia pestis, Francisella tularensis, Bacillus brucellae and five kinds of drastic pathogenic bacterias of pseudoglanders burkholderia, the invention still further relates to the method for utilizing described gene liquid chip to detect.
Background technology
The strong pathogenic agent that may be used for bio-terrorism has a variety of, comprises Bacillus anthracis, yersinia pestis, francisella tularensis, Bacillus brucellae, glanders burkholderia, pseudoglanders Burkholder bacillus, hemorrhagic fever virus, botulinus toxin and Ricin etc.
Existing pathogen detection technology all sees the detection of the bio-terrorism factor as biosensor or colloidal gold technique etc.These technology are sensitive or special, but are subject to multiple detectivity, are difficult to suspicious sample is carried out the multiplefactor screening.Suspending chip (suspension array) also claim liquid-phase chip (liquid array, liquid chip), combine the sensitivity of round pcr and the specificity of Nucleic Acid Probe Technique, and in a reacting hole, can detect 100 kinds of different PCR products, detection time is short, flux is big, examination when can realize the many targets of doubtful sample.
Bacillus anthracis (Bacillus anthracis) is the pathogenic agent that the people beast suffers from transmissible disease-anthrax altogether, is thick Gram-positive bacillus, has that intensive is pathogenic, infectivity and a viability.Yersinia pestis (Yersinia pestis) is the pathogenic agent of the deadly infectious disease-plague of serious harm human health, Gram-negative oval bacillus, and its natural host is a rodent, propagates by the mouse flea.。Francisella tularensis (Francisella tularensis) is the pathogenic agent of acute infectious disease-tularaemia (tularemia), gram-negative coccobacillus, and strong toxicity is propagated fast.Bacillus brucellae (Brucella) is the pathogenic agent of gibraltar fever (brucellosic), the Gram-negative bacteria of one group of small club shape, and the occurring in nature resistibility is stronger.Pseudoglanders burkholderia (Burkholderia pseudomallei) is the pathogenic agent of Amphixenosis-pseudoglanders disease, gram negative bacilli, and the occurring in nature resistibility is stronger.
The multiple detection liquid-phase chip of Bacillus anthracis of the present invention, yersinia pestis, Francisella tularensis, Bacillus brucellae and pseudoglanders burkholderia, realize diversification, the high-throughput examination of important pathogen, for prevention with control the cross-border propagation that disseminates infection, tackle bio-terrorism, safeguard that the country and people's life and health safety has great importance.
Summary of the invention
The present invention relates to gene liquid chip of a kind of joint-detection Bacillus anthracis, yersinia pestis, Francisella tularensis, Bacillus brucellae and pseudoglanders burkholderia and preparation method thereof, this gene liquid chip can be used for detecting simultaneously Bacillus anthracis, yersinia pestis, Francisella tularensis, Bacillus brucellae and five kinds of pathogenic agent of pseudoglanders burkholderia.The invention still further relates to the method for utilizing described gene liquid chip to detect.
Existing pathogen detection technology is subject to the predicament of multiple detectivity, and many suspicious samples need carry out the examination of many targets, the objective of the invention is by a kind of five kinds of drastic pathogenic bacteria liquid-phase chips and its production and application that detect simultaneously are provided, for the rapid detection of five kinds of drastic pathogenic bacterias provides a kind of sensitivity, special, quick, reliable method.
Principle of work of the present invention is: select the specific gene fragment of five kinds of bacteriums, design is at the Auele Specific Primer of five kinds of bacteriums; Design is positioned at the specific probe of five kinds of bacteriums of amplification region then, the coding microball of the different numberings of probe coupling, preparing can be to above-mentioned five kinds of liquid-phase chip that bacterium detects simultaneously, when using, at first use above-mentioned primer that five kinds of bacteriums are carried out the multiplex PCR amplification, add amplification PCR products then, make the capture probe on the microballoon catch corresponding PCR product, react with streptavidin-phycoerythrin (SA-PE) again, (as LUMINEX 100, LUMINEX 200, Bio-plex by the LUMINEX detection system, LIQUICHIP etc.), realization is to the disposable detection of five kinds of bacteriums.
Liquid-phase chip of the present invention comprise coding microball, with coding microball link coupled capture probe, biotinylated primer, PCR reaction system, streptavidin-phycoerythrin (SA-PE).
PCR reaction system of the present invention comprises 10 * PCR damping fluid, dNTPs, archaeal dna polymerase (Taq enzyme), biotinylated primer, deionized water.
The used coding microball of the present invention can derive from companies such as LUMINEX, BIO-RAD, QIAGEN, GENE, can select for use arbitrary number as coding microball, for example is selected from the 044th, 042,032,025,034, No. 027 microballoon of above-mentioned company.
Primer of the present invention can adopt conventional biotechnology to screen, design or buy in well known to a person skilled in the art scope, primer sequence of the present invention can for example be: Bacillus anthracis (Bacillus anthracis) (abbreviation anthrax-bacilus): two pairs of primers, wherein a pair of karyomit(e), a pair of plasmid that is positioned at of being positioned at, sequence is as follows:
BA-1-F：5’-TGGACGCATACGAGACATAAT-3’
BA-1-R：5’-TGCTTTAGCGGTAGCAGAGG-3’
BA-2-F：5’-TTTCATAATCATGGATTTCCCG-3’
BA-2-R：5’-TTACCCAACATCATCTTCGCA-3’
Yersinia pestis (Yersinia pestis) (being called for short plague bacillus): be positioned at karyomit(e)
YP-F：5’-ACTCAATGTTGTGACGAGGATG-3’
YP-R：5’-TTACTTCTAATGCCATCAGGTAGC-3’
Bacillus brucellae (Brucella):
Bru-F：5’-TGGCTCGGTTGCCAATATCAA-3’
Bru-R：5’-GCGCTTGCCTTTCAGGTCTG-3’
Francisella tularensis (Francisella tularensis) (be called for short soil and draw bacterium): fopA
FT-F：5’-GGGCAAATCTAGCAGGTCAAG-3’
FT-R：5’-GCTGTAGTCGCACCATTATCCT-3’
Pseudoglanders burkholderia (Burkholderia pseudomallei) (following abbreviation pseudoglanders bacterium):
BP-F：5’-CGATCTCGTCAAGGTGTCGG-3’
BP-R：5’-CCCCAGTTCATCTGATACTTGC-3’
Biotinylation of the present invention is meant when primer is synthetic primer sequence 5 ' end is carried out mark with vitamin H.
In PCR system of the present invention, the right concentration of primer can be respectively 60-150nmol/L.
In above-mentioned PCR system, the concentration of dNTPs can be 0.05-0.5 ∏ mol/L.
In above-mentioned PCR system, the Taq enzyme concn can be 1-5U/30 μ l.
Preferred PCR reaction system is 30 μ l systems: 10*PCR damping fluid 3 μ l; Taq enzyme 2U; 2.5mmol/L Mg
2+0.2 the concentration of ∏ mol/L dNTP primers F T-F, FT-R is respectively 60-100nmol/L; The concentration of primer BP-F, BP-R is respectively 60-100nmol/L; The concentration of primer BA-1-F, BA-1-R, BA-2-F, BA-2-R, YP-F, YP-R is respectively 90-110nmol/L; The concentration of primer Bru-F, Bru-R is respectively 100-150nmol/L; Dna profiling 2 μ l; DdH
2O complements to 30 μ l.
The condition of PCR reaction: 94 ℃ 10 minutes; 94 ℃ of 30s, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 7 minutes.Above-mentioned reaction conditions is the amplification instrument optimized reaction conditions according to ABI 9700, adopts other instruments according to circumstances to adjust reaction conditions, and this is known in those skilled in the art.
Sequence capture probe of the present invention is as follows:
Anthrax-bacilus probe 1:5 '-GAAGAACGCAGGCTTAGATTGGT-3 '
Anthrax-bacilus probe 2:5 '-CTCGCTTTCATCGCATTTCTCCC-3 '
Plague bacillus probe: 5 '-AACAGTAAGCATCCAGTCGTTCATA-3 '
Bacillus brucellae probe: 5 '-TTACGCAGTCAGACGTTGCCTAT-3 '
Soil draws bacterium probe: 5 '-TGCTGGTTTAACATGGTTCTTTGG-3 '
Pseudoglanders bacterium probe: 5 '-AGGTCAATTTCCCGAACAAGACT-3 '
Above described probe 5 ends when synthetic all amido modified and connect 15-20 T as at interval, the described coding microball of coupling the present invention relates to a kind of preparation method of above-mentioned liquid-phase chip then, this method comprises:
(1) microballoon and capture probe coupling
(2) choose the coding microball of certain numbering respectively, for example described coding microball is selected from: 044,042,032,025, and 034, No. 027 microballoon; Washing, activation; Corresponding respectively anthrax-bacilus probe 1 and the probe 2 of adding, the plague bacillus probe, Bacillus brucellae probe, soil draw the bacterium probe, pseudoglanders bacterium probe; Hatch 30～60min under the mixing, room temperature; Repeat 1～2 time; Wash 1～2 time with PBST (being phosphoric acid salt-tween damping fluid); Wash 1～2 time with 0.1%SDS (being sodium laurylsulfonate); Microballoon is resuspended in the TE damping fluid, tally's concentration; 4 ℃ keep in Dark Place.
The present invention relates to utilize above-mentioned liquid-phase chip accurately to detect the detection method of above-mentioned five kinds of bio-terrorism bacterium simultaneously, this method comprises: 1, the extraction of sample nucleic acid to be detected, 2, carry out multi-PRC reaction, wherein prepare certain concentration of component, certain volume is the PCR reaction system of 30 μ l for example, 3, capture probe is caught the PCR product: coupling is had the microballoon of probe mix with the multiple PCR products of biotinylation mark, 95 ℃ of sex change 5～10 minutes, hybridized 10～30 minutes for 45～60 ℃, probe can specificity be caught corresponding PCR product, and then adds streptavidin-phycoerythrin.
In the extraction of the sample nucleic acid to be detected of the first step, the extraction of sample nucleic acid to be checked can be adopted the phenol-chloroform method for extracting, perhaps uses general commercial kit to extract, and perhaps adopts NaI (being sodium iodide) cracking-glass powder absorption method.
In above-mentioned multi-PRC reaction, most preferably according to following concentration configuration PCR reaction system, 30 μ l systems: 10*PCR damping fluid 3 μ l; Taq enzyme 2U; 2.5mmol/L Mg
2+0.2 ∏ mol/L dNTP; Each 80nmol/L of FT-F, FT-R, each 80nmol/L of BP-F, BP-R, BA-1-F, BA-1-R, BA-2-F, BA-2-R, each 100nmol/L of YP-F, YP-R, each 120nmol/L of Bru-F, Bru-R; In the PCR pipe, add template 2 μ l, add ddH again
2O supplies 30 μ l.Reaction conditions: 94 ℃ 10 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ extend to 7 minutes.
Catch in the step of PCR product at capture probe, coupling there is the microballoon of probe mix with the multiple PCR products of biotinylation mark, 95 ℃ of sex change 5～10 minutes, hybridized 10～30 minutes for 45～60 ℃, probe can specificity be caught corresponding PCR product, and then adding streptavidin-phycoerythrin, the reporter molecules that has fluorescence then combines with vitamin H, this complex body is by the suspending chip detection system, by red, green two bundle laser detection, thereby red laser excites the dyestuff of microballoon matrix that the microballoon numbering is discerned, and green laser is discerned surperficial bonded fluorescence dye, realizes the quantitative analysis of the PCR product that capture probe is caught.
Description of drawings
Accompanying drawing is a DNA concentration---average fluorescent strength (MFI) dose response curve on coding microball surface, and X-coordinate is a concentration: fg/ reaction system, ordinate zou are the MFI value, wherein:
Fig. 1 is Francisella tularensis DNA concentration-MFI dose response curve;
Fig. 2 is Bacillus brucellae (M5 strain) DNA concentration-MFI dose response curve;
Fig. 3 is DNA concentration-MFI dose response curve that Bacillus anthracis probe 1 detects;
Fig. 4 is DNA concentration-MFI dose response curve that Bacillus anthracis probe 2 detects;
Fig. 5 is pseudoglanders burkholderia DNA concentration-MFI dose response curve;
Fig. 6 is yersinia pestis (EV76 strain) DNA concentration-MFI dose response curve.
Embodiment
The present invention is to detect the detection method of five kinds of bio-terrorism bacterium, and the invention will be further described below by embodiment, but the present invention is subjected to the qualification of the following example never in any form.
Embodiment 1: the multi-PRC reaction that adopts six pairs of primers of five kinds of bacteriums
1. the extraction of sample amplifying nucleic acid: NaI cracking-glass powder absorption method is extracted nucleic acid.
2. prepare the multi-PRC reaction system by following system, cumulative volume 30 μ l:
10×PCR?buffer： 3μl
Taq?E： 0.4μl
dNTPs： 0.6μl
BA-1-F, BA-1-R (10 ∏ M): each 0.3 ∏ l
BA-2-F, BA-2-R (10 ∏ M): each 0.3 ∏ l
YP-F, YP-R (10 ∏ M): each 0.3 ∏ l
Bru-F, Bru-P (10 ∏ M): each 0.36 ∏ l
FT-F, FT-R (10 ∏ M): each 0.24 ∏ l
BP-F, BP-R (10 ∏ M): each 0.24 ∏ l
Template DNA: 2 ∏ l
DdH
2O: add to 30 ∏ l
3. carry out the PCR reaction by following reaction conditions
Pre-sex change:
94 ℃ 10 minutes
32 circulations:
94 ℃ 30 seconds
58 ℃ 30 seconds
72 ℃ 40 seconds
Extend:
72 ℃ 10 minutes
Reaction finishes the back with 1% agarose gel electrophoresis inspection amplification situation.
PCR negative control: except that not adding template and with ddH
2Outside O replaced, all the other systems were identical.
Embodiment 2: capture probe and microballoon coupling
1. choose 044,042,032,025 respectively, 034, No. 027 coding microball with vortex oscillation device vibration microballoon suspension, mixes microballoon.
2. get above-mentioned microballoon about 1.25 * 10 respectively
6Individual, be transferred to centrifuge tube respectively, the centrifugal 3-5Min of 14000g, careful sucking-off supernatant.
3. 2-(n-morpholino) the ethyl sulfonic acid solution that adds 50 ∏ l 0.1mol/L, vibration 20-30S, ultrasonic 20-30s makes microballoon resuspended.
4. with distilled water the synthetic oligonucleotide probe is diluted to 0.1mmol/L.
5. the probe with 1 ∏ l dilution is added in the microsphere suspension liquid, and corresponding to 044,042,032,025,034, No. 027 coding microball adds anthrax-bacilus probe 1 respectively, probe 2, and the plague bacillus probe, Bacillus brucellae probe, soil draw the bacterium probe, pseudoglanders bacterium probe, vibration mixing.
6. the EDC solution that adds the freshly prepared 10mg/mL of 2.5 ∏ l is to microballoon and the mixed liquid of probe, and mixing vibrates.
7. with aluminium foil parcel centrifuge tube lucifuge, 400-600rpm vibration on the vortex oscillation device, incubated at room 30 minutes.
8. add freshly prepared 10mg/mL EDC once more.
9. 400-600rpm vibration on the vortex oscillation device once more, the room temperature lucifuge was hatched 30 minutes.
10. with 0.02%PBST 1ml washing 1 time, centrifugal 14000g 3-5 minute.
Abandon supernatant 11. move, microballoon is resuspended among the 1ml 0.1%SDS, and washing is centrifugal.
Abandon supernatant 12. move, microballoon is resuspended among the 100 ∏ l pH8.0TE, and mixing has been hanged in vibration, promptly obtains the good detection microballoon of coupling.
13., converse the unit concentration of every kind of microballoon with the quantity of Hematocyte Counter counting microballoon.
Keep in Dark Place 14. the good detection microballoon of coupling is placed on 4 ℃, general every kind of probe link coupled microballoon is preserved separately, during use, selects to want blended microballoon kind according to test item.
Embodiment 3: the liquid-phase chip with above-mentioned five kinds of drastic pathogenic bacterias detects sample to be tested
1. get each 3500 mixing of above-mentioned various coupling microballoon, be sub-packed in the PCR pipe and (calculate corresponding add-on) according to the microballoon count results
2. it is 50 ∏ l that the PCR product that adds five kinds of bacterium hybrid templates of 5～17 ∏ l in each pipe makes its final volume, the piping and druming mixing.
3.95 ℃ sex change 10 minutes.
4.45～60 ℃ were reacted 10～30 minutes.
5. be transferred to 96 hole filter plate suction filtrations and remove unconjugated PCR product.
6. add 75 ∏ l4ng/ ∏ lSA-PE to each hole again, the room temperature lucifuge was hatched 10 minutes, and suction filtration removes unconjugated SA-PE.
7. add 75 ∏ l suspension to each hole again, vibration makes microballoon resuspended.
8. reaction finishes upward machine testing of back.
9. suspending chip detection system, by red, green two bundle laser detection, thereby red laser excites the dyestuff of microballoon matrix that the microballoon numbering is discerned, and green laser is discerned surperficial bonded fluorescence dye, realizes the quantitative analysis of the PCR product that capture probe is caught.
Following table is the suspending chip detected result of above-mentioned five kinds of pathogenic bacterias to be measured.
In the table: Type is a sample type, and B represents negative control, and C represents known sample, and X represents unknown sample; BA-1 is a Bacillus anthracis probe 1, BA-2 is a Bacillus anthracis probe 2, the negative contrast of BSA, Yp-2 is the yersinia pestis probe, Bru-2 is the Bacillus brucellae probe, Ft-2 is the Francisella tularensis probe, and Bp-2 is a pseudoglanders burkholderia probe, and Biotin (045) is the vitamin H positive control; C1, C2, C4 are the Bacillus anthracis sample, and C6, C7, C8 are the yersinia pestis sample, and C9, C10 are the Bacillus brucellae sample, the negative sample of C11; Detect fluorescent value and be judged to the positive more than or equal to three times of background fluorescence values, thus all correct identification of known sample, unknown sample X1, X2 are the Bacillus anthracis strong positive, and the pseudoglanders Burkholder is weak positive.
Accompanying drawing is depicted as the fluorescence intensity dose response curve on DNA concentration-coding microball surface.
As can be seen from the figure coding microball surface fluorescence intensity becomes positive correlation with the template amount of adding in certain scope, the amount of the template DNA that adds in the pipe when wherein X-coordinate is multiplex PCR, and ordinate zou is represented the fluorescence intensity of corresponding encoded microsphere surface.Can the typical curve by match realize semi-quantitative analysis to nucleic acid.
Conclusion:
Can draw from The above results, suspending chip of the present invention and detection method have following superiority:
1. sensitive: compare to common multi-PRC reaction, sensitivity of the present invention improves from following two The aspect embodies: 1) there is the amplification system of signal in detecting instrument; 2) biotin on the PCR product band The amplification of being combined with the Avidin that the fluorescent dye phycoerythrin connects.
2. special: the employing Nucleic Acid Probe Technique is carried out specific knowledge to the PCR product of biotin on the mark Not, and unconventional electrophoresis method is identified by PCR product clip size.
3. high flux: detect when one time PCR reaction one-time detection can realize the plurality of target bacterium. 4. precise and high efficiency: integrate nucleic acid amplification in vitro, making nucleic acid molecular hybridization, coding microball, biotin mark Note, fluoroscopic examination and Flow Cytometry are realized detection, the evaluation of nucleic acid samples simultaneously in one, Make the result accurate, efficient work.
5, chip manufacturing is convenient: only with the primer that designs object bacteria to be checked, and optimize PCR condition, mark The probe of note correspondence namely can be assembled into detection architecture in coding microball.
6, the Multiple detection target is flexibly adjustable: can detect target according to difference, select corresponding primer and little Ball forms the detection architecture that different target makes up.
Claims (13)
1, the method for the multiple pathogenic agent of a kind of joint-detection, it is characterized in that, this method is used liquid-phase chip, described chip comprises microballoon, capture probe, biotinylated primer, PCR reaction system, streptavidin-phycoerythrin (SA-PE), and described pathogenic agent is Bacillus anthracis, yersinia pestis, Francisella tularensis, Bacillus brucellae and pseudoglanders Bock Hall moral bacterium.
2, detection method as claimed in claim 1 is characterized in that, described PCR reaction system comprises 10 * PCR damping fluid, dNTPs, archaeal dna polymerase, biotinylated primer, deionized water.
3, detection method as claimed in claim 1 is characterized in that, the primer sequence that this method is used is as follows:
Bacillus anthracis (Bacillus anthracis): two pairs of primers, the wherein a pair of a pair of plasmid that is positioned at of karyomit(e) that is positioned at, sequence is as follows:
BA-1-F：TGGACGCATACGAGACATAAT
BA-1-R：TGCTTTAGCGGTAGCAGAGG
BA-2-F：TTTCATAATCATGGATTTCCCG
BA-2-R：TTACCCAACATCATCTTCGCA
Yersinia pestis (Yersinia pestis): be positioned at karyomit(e), sequence is as follows,
YP-F：ACTCAATGTTGTGACGAGGATG
YP-R：TTACTTCTAATGCCATCAGGTAGC
Bacillus brucellae (Brucella): sequence is as follows,
Bru-F：TGGCTCGGTTGCCAATATCAA
Bru-R：GCGCTTGCCTTTCAGGTCTG
Francisella tularensis (Francisella tularensis): fopA, sequence is as follows,
FT-F：GGGCAAATCTAGCAGGTCAAG
FT-R：GCTGTAGTCGCACCATTATCCT
Pseudoglanders Bock Hall moral bacterium (Burkholderia pseudomallei): sequence is as follows
BP-F：CGATCTCGTCAAGGTGTCGG
BP-R：CCCCAGTTCATCTGATACTTGC。
4, the described detection method of claim 1 is characterized in that, sequence capture probe used in this method is as follows:
Anthrax-bacilus probe 1:GAAGAACGCAGGCTTAGATTGGT
Anthrax-bacilus probe 2:CTCGCTTTCATCGCATTTCTCCC
Plague bacillus probe: AACAGTAAGCATCCAGTCGTTCATA
Bacillus brucellae probe: TTACGCAGTCAGACGTTGCCTAT
Soil draws bacterium probe: TGCTGGTTTAACATGGTTCTTTGG
Pseudoglanders bacterium probe: AGGTCAATTTCCCGAACAAGACT
5, as each described detection method of claim 1-4, it is characterized in that, the method comprising the steps of: 1, the extraction of sample nucleic acid to be detected, 2, multi-PRC reaction, prepare 30 μ l PCR reaction systems of certain concentration of component, 3, capture probe is caught the PCR product: coupling is had the microballoon of probe mix with the multiple PCR products of biotinylation mark, 95 ℃ of sex change 5～10 minutes, hybridized 10～30 minutes for 45～60 ℃, probe can specificity be caught corresponding PCR product, and then adds streptavidin-phycoerythrin.
6, detection method as claimed in claim 1 is characterized in that, when primer is synthetic sequence 5 ' end is carried out biotin labeling.
As each described detection method of claim 1-4, it is characterized in that 7, in described PCR system, the right concentration of primer is respectively 60-150nmol/L.
8, detection method as claimed in claim 7 is characterized in that, in described PCR system, the concentration of dNTPs is 0.05-0.5 ∏ mol/L.
As the described detection method of claim 1-4, it is characterized in that 9, in described PCR system, the Taq enzyme concn is 1-5U/30 μ l.
10, detection method as claimed in claim 5 is characterized in that, the PCR reaction system is 30 μ l systems: 10*PCR damping fluid 3 μ l; Taq enzyme 2U; 2.5mmol/L Mg
2+0.2 the concentration of ∏ mol/L dNTP primers F T-F, FT-R is respectively 60-100nmol/L; The concentration of primer BP-F, BP-R is respectively 60-100nmol/L; The concentration of primer BA-1-F, BA-1-R, BA-2-F, BA-2-R, YP-F, YP-R is respectively 90-110nmol/L; The concentration of primer Bru-F, Bru-R is respectively 100-150nmol/L; Dna profiling 2 μ l; DdH
2O supplies 30 μ l.
11, detection method as claimed in claim 5 is characterized in that, the condition of described PCR reaction: 94 ℃ 10 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 7 minutes.
As each described detection method of claim 1-4, it is characterized in that 12, the PCR reaction system is 30 μ l systems: 10*PCR damping fluid 3 μ l; Taq enzyme 2U; 2.5mmol/L Mg
2+0.2 the concentration of ∏ mol/L dNTP primers F T-F, FT-R is respectively 60-100nmol/L; The concentration of primer BP-F, BP-R is respectively 60-100nmol/L; The concentration of primer BA-1-F, BA-1-R, BA-2-F, BA-2-R, YP-F, YP-R is respectively 90-110nmol/L; The concentration of primer Bru-F, Bru-R is respectively 100-150nmol/L; Dna profiling 2 μ l; DdH
2O supplies 30 μ l.
13, a kind of method for preparing the liquid phase probe of the strong pathogenic agent of each described detection of claim 1-4 is specially:
Choose the coding microball of certain numbering respectively; Washing, activation; Corresponding respectively anthrax-bacilus probe 1 and the probe 2 of adding, the plague bacillus probe, Bacillus brucellae probe, soil draw the bacterium probe, pseudoglanders bacterium probe; Hatch 30～60min under the mixing, room temperature; Repeat 1～2 time; Wash 1～2 time with PBST (being phosphoric acid salt-tween damping fluid); Wash 1～2 time with 0.1%SDS (being sodium laurylsulfonate); Microballoon is resuspended in the TE damping fluid, tally's concentration; 4 ℃ keep in Dark Place.
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