CN101629211B - Improved gene liquid chip for detecting bacillus anthracis and preparation method and application thereof - Google Patents
Improved gene liquid chip for detecting bacillus anthracis and preparation method and application thereof Download PDFInfo
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- CN101629211B CN101629211B CN2009100788220A CN200910078822A CN101629211B CN 101629211 B CN101629211 B CN 101629211B CN 2009100788220 A CN2009100788220 A CN 2009100788220A CN 200910078822 A CN200910078822 A CN 200910078822A CN 101629211 B CN101629211 B CN 101629211B
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Abstract
The invention relates to a gene liquid chip for detecting bacillus anthracis and a preparation method thereof. The invention also relates to a method adopting the gene liquid chip for detection. The invention establishes the liquid chip for detecting the bacillus anthracis and the detection method by taking pXO2 and specific genes on the chromosomes as target sequences, thus providing a feasible way for detecting the bacillus anthracis.
Description
Technical field
The present invention relates to a kind of gene liquid chip that detects Bacillus anthracis and preparation method thereof.The invention still further relates to the method for utilizing described gene liquid chip to detect.
Background technology
It is the emerging technology that grew up in recent years that liquid-phase chip detects, and has quick, special, responsive characteristics.The ultimate principle of suspending chip is to utilize the microballoon of PS (polystyrene) made; Coat the ruddiness and the infrared light chromogenic reagent of different ratios; And produce 100 kinds of different ratios colors, and as the color numbers of 100 kinds of uniquenesses, every about 5.5 μ m of microballoon size; Can study purposes such as immunoassay, nucleic acids research, enzyme analysis, acceptor and part discriminance analysis etc. according to different, and demarcate antibodies specific, nucleic probe and various acceptor probe according to difference research purpose.
Bacillus anthracis (Bacillus anthracis) is the pathogenic agent of acute infectious disease anthrax, is thick Gram-positive bacillus, has that intensive is pathogenic, infectivity and a viability.The mankind can cause skin, lung and intestinal tract infections through direct or indirect contact anthrax bacillus, cause malignant pustule, anthrax pneumonia and enteron aisle anthrax.Anthrax bacillus has two toxin plasmid of being correlated with, the pXO2 plasmid (96kb) of the pXO1 plasmid (181kb) of the anthrax toxin of promptly encoding and coding pod membrane synthetic enzyme.PXO1 and pXO2 are necessary to toxicity, lose any one plasmid and all will weaken toxicity or make it become avirulent strain.The virulence of anthrax bacillus depends primarily on the ability that produces pod membrane and toxin.
At present, the laboratory diagnostic method of Bacillus anthracis mainly is to lean on smear microscopy, cultivation proterties, phage test etc., and these methods are used to detect non-clinical sample length consuming time, and will possess corresponding techniques quality.
Summary of the invention
The present invention relates to a kind of improved gene liquid chip and preparation method thereof, this gene liquid chip can be used for the detection of pathogenic agent Bacillus anthracis.The invention still further relates to the method for utilizing described gene liquid chip to detect.
Through deeply with a large amount of research and experiments, the present invention is a target sequence with the specific gene on pXO2 and the karyomit(e), has set up Bacillus anthracis liquid-phase chip and detection method, for the detection of Bacillus anthracis provides a feasible approach.
Gene liquid chip of the present invention comprises: microballoon, capture probe, primer, PCR reaction system, streptavidin-phycoerythrin, this gene liquid chip can detect Bacillus anthracis.
The invention still further relates to the gene liquid chip of above-mentioned detection Bacillus anthracis, this chip comprises: fluorescence-encoded microballoon, capture probe, primer, PCR reaction system, streptavidin-phycoerythrin, wherein
(1) the PCR reaction system comprises: 10 * PCR damping fluid, dNTPs, Taq archaeal dna polymerase.
(2) primer comprises:
Wherein, downstream primer is 5, biotin labeled BA-1-R, and BA-2-R,
Specifically be BA-1-R:5 '-vitamin H-TGCTTTAGCGGTAGCAGAGG
BA-2-R:5 '-vitamin H-TTACCCAACATCATCTTCGCA
(3) capture probe, described capture probe has specificity:
| The probe title | The probe target position | Probe sequence (5 '-3 ') |
| BA-1 | The bp415-437 of Bacillus anthracis str.A2012 plasmid pXO2 capB | GAAGAACGCAGGCTTAGATTGGT |
| BA-2 | The bp742-764 of Bacillus anthracis str. ' AmesAncestor ' AE017334 | CTCGCTTTCATCGCATTTCTCCC |
Above said probe when synthetic, carry out amido modifiedly at 5 ' end, and connect 15-20 T or connection C12 as spacer chain, then probe and selected coding microball are carried out coupling.
(4) microballoon is generally selected fluorescence-encoded microballoon, for example derives from the microballoon of any numbering of Luminex or other shipping agencies, and specific probe and fluorescence-encoded micro-beads coupling form and detect microballoon, constitute detection chip thus.
(5) (Streptavidin-R-phycoerythrin SA-PE) is a kind of GFP to streptavidin-phycoerythrin, can be carried out qualitative or detection by quantitative thus by the green laser stimulated luminescence in the liquid-phase chip detection system.
The present invention relates to the preparation method of said gene liquid-phase chip, this method comprises:
1. choose coding microball, get in right amount according to appointment 1.25 * 10
6Individual in centrifuge tube, centrifugal abandon supernatant after, 2-(n-morpholino) the ethyl sulfonic acid solution that adds 0.1mol/L makes it resuspended;
2. with zero(ppm) water synthetic oligonucleotide probe (being capture probe 1 and 2) is diluted, add to respectively in two kinds of microsphere suspension liquids, the vibration mixing;
3. add in EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) solution to the microballoon and the mixed liquid of probe of 10mg/mL vibration mixing, incubated at room 30 minutes;
4. with the 0.02%PBST washing, centrifugal under the 14000g; Move and abandon supernatant, above-mentioned microballoon is resuspended among the 1ml 0.1%SDS (being sodium laurylsulfonate), washing, centrifugal;
5. move and abandon supernatant, microballoon is resuspended among 100 ∏, the 1 pH8.0 TE, and mixing has been hanged in vibration, obtains the good detection microballoon of coupling, gene liquid chip promptly of the present invention.4 ℃ keep in Dark Place.
On the other hand, the invention still further relates to the detection method that above-mentioned Bacillus anthracis detects gene liquid chip, this method comprises: the extraction of sample nucleic acid, pcr amplification detects pathogenic agent.The present invention improves the operation of above-mentioned three steps and condition and optimizes.
The extraction of sample nucleic acid:
Adopt NaI cracking-glass powder absorption method to extract the nucleic acid of sample.Specific as follows:
1) in every pipe sample, add 6mol/L NaI, the vibration mixing boils;
2) centrifugal, change supernatant and contain in the centrifuge tube of 20% glass powder liquid to another;
3) abundant mixing, room temperature was placed 10 minutes, and the DNA of exposure is adsorbed on the glass powder; 8000r/ minute centrifugal, and supernatant carefully inclines;
4) in glass powder-DNA deposition, add 75% ethanol 1ml, abundant mixing, centrifugal, supernatant carefully inclines;
5) repeating step (4) places 37 ℃ of thermostat container inner dryings then;
6) in above-mentioned exsiccant glass powder-DNA deposition, add 20 μ l TE, abundant mixing, 65 ℃ of heating, centrifugal, it is subsequent use to reclaim supernatant.
Pcr amplification:
With the template that the supernatant for preparing in the above-mentioned nucleic acid extraction step reacts as PCR, PCR reaction system and reaction conditions:
The PCR system:
10 * PCR damping fluid: 3 ∏ 1
Taq archaeal dna polymerase: 0.3 ∏ 1
dNTPs： 0.3∏1
Upstream primer: 0.3 ∏ 1
Downstream primer: 0.3 ∏ 1
Template DNA: 2 ∏ 1
Distilled water ddH
2O: supply 30 ∏ 1
Reaction conditions:
Sex change in advance:
94-96 ℃ 10 minutes
35 circulations:
94-96 ℃ of 30-40 second
55-58 ℃ of 30-40 second
72 ℃ of 30-40 seconds
Extend:
72 ℃ 4-7 minute
Simultaneously, PCR blank article are as follows:
10 * PCR damping fluid: 3 ∏ 1
Taq enzyme: 0.3 ∏ 1
dNTPs： 0.3∏1
Upstream primer: 0.3 ∏ 1
Downstream primer: 0.3 ∏ 1
Distilled water ddH
2O: supply 30 ∏ 1
Reaction finishes the back and checks the amplification situation with 1% agarose gel electrophoresis.
Detection method:
1) gets above-mentioned two kinds of coupling microballoons and mix, be loaded in the PCR pipe, calculate corresponding add-on, make the amount of every kind of microballoon identical, react every kind of 3000-5000 at every turn according to the microballoon count results;
2) in each pipe, add the product that the pcr amplification above 5～17 ∏ 1 obtains, mixing;
3) 95 ℃ of sex change are 10 minutes; 45～60 ℃ were reacted 10～30 minutes subsequently;
4) centrifugal or suction filtration removes unconjugated PCR product;
5) add SA-PE to each hole again, the room temperature lucifuge is hatched, and suction filtration removes unconjugated SA-PE;
6) add 75 ∏ 1TE suspension-s to each hole again, vibration makes microballoon resuspended;
7) going up the LUMINEX detecting instrument after reaction finishes detects.For example use the Bio-plex test set; Through 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 the GFP of combination, realizes the quantitative analysis of the PCR product that capture probe is caught.
Description of drawings:
Fig. 1: detect the dose-response curve of Bacillus anthracis nucleic acid with probe 1, i.e. corresponding relation between the amount of template DNA and the fluorescent signal value MFI that reacts; Transverse axis is represented the concentration (fg/test) of template DNA in the PCR reaction system, and the longitudinal axis is represented fluorescent signal value MFI.
Fig. 2: the dose-response curve that detects Bacillus anthracis nucleic acid with probe 2; Transverse axis is represented the concentration (fg/test) of template DNA in the PCR reaction system, and the longitudinal axis is represented fluorescent signal value MFI.
Embodiment
The preparation of embodiment 1, gene liquid chip
1. choose coding microball,, get about 1.25 * 10 as 044, No. 042
6The individual centrifuge tube that places, centrifugal under the 14000g, for example centrifugal 3-5 minute, careful sucking-off supernatant;
2. 2-(n-morpholino) the ethyl sulfonic acid solution that adds 50 ∏, 1 0.1mol/L, the vibration mixing.
3. with zero(ppm) water the synthetic oligonucleotide probe is diluted to 0.1mmol/L; The probe 1 and 2 of 1 ∏ 1 dilution is added on respectively in 044, No. 042 microsphere suspension liquid, and mixing vibrates.
4. add in EDC solution to the microballoon and the mixed liquid of probe of 2.5 ∏, 1 freshly prepared 10mg/mL vibration, incubated at room 30 minutes.
5. with 0.02%PBST 1ml washing 1 time, centrifugal 14000g 3-5 minute.
6. move and abandon supernatant, microballoon is resuspended among the 1ml 0.1%SDS, and washing is centrifugal.
7. move and abandon supernatant, microballoon is resuspended among 100 ∏, the 1 pH8.0 TE, and mixing has been hanged in vibration, promptly obtains the good detection microballoon of coupling.
8. with the quantity of Hematocyte Counter counting microballoon, converse the unit concentration of every kind of microballoon.
9. be placed on 4 ℃ to the good detection microballoon of coupling and keep in Dark Place, general every kind of probe link coupled microballoon
Preserve separately, during use, select to want blended microballoon kind according to test item.
The extraction of embodiment 2, sample nucleic acid
Adopt NaI cracking-glass powder absorption method to extract the nucleic acid of sample.Specific as follows:
1) in every pipe sample, add 6mol/L NaI 100 μ l, the vibration mixing boiled 30 minutes.
2) with the above-mentioned sample that boils processing centrifugal 5 minutes, supernatant is gone in another centrifuge tube that contains 20% glass powder liquid in 10000r/ minute.
3) abundant mixing was put room temperature 10 minutes, and 8000r/ minute centrifugal 2 minutes, supernatant carefully inclined.
4) in glass powder DNA deposition, add 75% ethanol 1ml, abundant mixing, the cleaning glass powder, 8000r/ minute centrifugal 2 minutes, supernatant carefully inclined.
5) repeated washing once after, be positioned over 37 ℃ of thermostat containers and make finish-drying.
6) in above-mentioned air dried glass powder-DNA deposition, add 20 μ lTE, abundant mixing, 65 ℃ were heated 15 minutes.8000r/ minute centrifugal 2 minutes, it was subsequent use to reclaim supernatant.
Embodiment 3, pcr amplification
With the template that the supernatant that reclaims reacts as PCR, PCR reaction system and reaction conditions:
The PCR system:
10 * PCR damping fluid: 3 ∏ 1
Taq enzyme: 0.3 ∏ 1
dNTPs： 0.3∏1
Upstream primer: 0.3 ∏ 1
Downstream primer: 0.3 ∏ 1
Template DNA: 2 ∏ 1
Distilled water ddH
2O: supply 30 ∏ 1
Reaction conditions:
Sex change in advance:
94 ℃ 10 minutes
35 circulations:
94 ℃ 30 seconds
58 ℃ 30 seconds
72 ℃ 40 seconds
Extend:
72 ℃ 7 minutes
The PCR blank is set simultaneously, as follows:
10 * PCR damping fluid: 3 ∏ 1
Taq enzyme: 0.3 ∏ 1
dNTPs： 0.3∏1
Upstream primer: 0.3 ∏ 1
Downstream primer: 0.3 ∏ 1
Distilled water ddH
2O: supply 30 ∏ 1
Reaction finishes the back and checks the amplification situation with 1% agarose gel electrophoresis.
Embodiment 4, detection
1. get 042, No. 044 each 5000 mixing of coupling microballoon, be sub-packed in the PCR pipe (calculating corresponding add-on) according to the microballoon count results
2. in each pipe, adding PCR product above 5～17 ∏ 1, to make its final volume be 50 ∏ 1.
3.95 ℃ sex change 10 minutes.
4.45～60 ℃ were reacted 10～30 minutes.
5. be transferred to the filter plate suction filtration and remove unconjugated PCR product.
6. add 75 ∏, 1 4ng/ ∏, 1 SA-PE to each hole again, the room temperature lucifuge was hatched 10 minutes, and suction filtration removes unconjugated SA-PE.
7. add 75 ∏, 1 suspension-s to each hole again, vibration makes microballoon resuspended.
8. reaction finishes upward machine testing of back.
9.Bio-plex detection system; Through red, green two bundle laser detection; Thereby red laser excites the dyestuff of microballoon matrix that the microballoon numbering is discerned, and green microballoon is discerned surperficial bonded optical dye, realizes the quantitative analysis of the PCR product that capture probe is caught.
When detecting, detect as detecting background with the PCR negative control simultaneously.For each detection architecture and detection background; The data of instrument output are a kind of fluorescence intensity median (Median Fluorescence Intensity that numbers population of microspheres in the respective reaction system; That is the statistical average value of each microballoon strength of signal of the population of microspheres of this numbering that reads (100 or more than) MFI).The result judges: when the MFI of sample aperture to be detected value is that this detection background strength of signal is judged to the positive more than three times the time.
Embodiment 5, detection by quantitative:
Extract the genomic dna of Bacillus anthracis, utilize its concentration of nucleic acid-protein analysis-e/or determining, carry out serial dilution, behind the pcr amplification, above detects step and detects, and Bio-Plex Version4.0 analyzes, and simulates dose-response curve and curvilinear equation.Pattern detection value substitution equation is realized detection by quantitative.
The result
To the detection of Bacillus anthracis, the present invention has designed two probes that are positioned at karyomit(e) and plasmid respectively and has detected, i.e. probe 1 and probe 2.Bacillus anthracis virulent strain 17003-10 genomic dna carries out 10 times of serial dilutions after measuring concentration, and 16fg～160ng carries out pcr amplification and detection by above-mentioned definite method, and matched curve draws curvilinear equation:
The curvilinear equation of probe 1: FI=-9.77647+ (1527.96+9.77647)/[(1+ (Conc/1.23743E+006)
-1.05785]
0.774541
The curvilinear equation of probe 2: FI=-5.51506+ (3841.16+5.51506)/[(1+ (Conc/280994)
-0.954295)]
1.44612
Calculate detection limit from typical curve, when template concentrations was 30pg/test in the PCR reaction tubes, probe 1 signal was positive; When template concentrations was 17pg/test in the PCR reaction tubes, probe 2 signals were positive.Therefore, to plasmid pXO2 male Bacillus anthracis virulent strain, detecting of body series is limited to 30pg/test; And for the negative strain of plasmid pXO2, detect and be limited to 17pg/test.
Can draw from The above results, suspending chip of the present invention and detection method have following meliority:
1. sensitive: compare to common multi-PRC reaction, sensitivity of the present invention improves from the embodiment of following two aspects: 1) there is the amplification system of signal in detecting instrument; 2) the avidin bonded amplification that is connected with the optical dye phycoerythrin of the vitamin H on the PCR product band.
2. special: the employing Nucleic Acid Probe Technique is carried out specific identification to the PCR product of vitamin H on the mark, and non-traditional electrophoresis method is discerned through PCR product clip size.
3. precise and high efficiency: integrate nucleic acid amplification in vitro, making nucleic acid molecular hybridization, coding microball, biotin labeling, fluoroscopic examination and Flow Cytometry in one, realize detection, the evaluation of nucleic acid samples simultaneously, make the result accurate, efficient work.
4, chip manufacturing is convenient: only with the primer that designs object bacteria to be checked, optimize the PCR condition, the probe of mark correspondence promptly can be assembled into detection architecture in coding microball.
5, the method that the present invention relates to is equally applicable to the detection of nucleic acids of other pathogenic agent or target compound.
6, can realize multiple detection, target to be checked is adjustable flexibly: can detect target according to difference, select corresponding primer and microballoon, form the detection architecture of different target combination.
Sequence table
< 110>China Inst. of Quarantine Inspection Sciences
< 120>gene liquid chip of a kind of improved detection Bacillus anthracis
The foundation of < 130>five kinds of bio-terrorism bacterial gene suspending chip multiple detection methods
<160>6
<170>Patentln?version?3.3
<210>1
<211>21
<212>DNA
< 213>upstream primer-1
<400>1
tggacgcata?cgagacataa?t 21
<210>2
<211>22
<212>DNA
< 213>upstream primer-2
<400>2
tttcataatc?atggatttcc?cg 22
<210>3
<211>20
<212>DNA
< 213>downstream primer-1
<400>3
tgctttagcg?gtagcagagg 20
<210>4
<211>21
<212>DNA
< 213>downstream primer-2
<400>4
ttacccaaca?tcatcttcgc?a 21
<210>5
<211>23
<212>DNA
< 213>probe-1
<400>5
gaagaacgca?ggcttagatt?ggt 23
<210>6
<211>23
<212>DNA
< 213>probe-2
<400>6
ctcgctttca?tcgcatttct?ccc 23
Claims (1)
1. gene liquid chip system that detects Bacillus anthracis, this chip system comprises:
No. 042 fluorescence-encoded microballoon of Luminex company, capture probe, primer, PCR reaction system, streptavidin-phycoerythrin;
Wherein said primer comprises:
BA-2-F?TTTCATAATCATGGATTTCCCG
BA-2-R?TTACCCAACATCATCTTCGCA
Downstream primer 5 ' is held through biotin labeling:
BA-2-R:5 '-vitamin H-TTACCCAACATCATCTTCGCA;
Wherein said capture probe is
Probes probes sequence (5 '-3 ')
Title
BA-2CTCGCTTTCATCGCATTTCTCCC
Above said probe when synthetic, carry out amido modifiedly at 5 ' end, and connect 15-20 T or connection C12 as spacer chain, then No. 042 fluorescence-encoded microballoon of probe and selected Luminex company is carried out coupling.
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| CN2009100788220A CN101629211B (en) | 2009-03-04 | 2009-03-04 | Improved gene liquid chip for detecting bacillus anthracis and preparation method and application thereof |
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| CN101629211B true CN101629211B (en) | 2012-07-25 |
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| CN102146470B (en) * | 2011-02-24 | 2013-02-20 | 广州华峰生物科技有限公司 | Bacillus anthracis detection kit and using method of kit |
Citations (2)
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|---|---|---|---|---|
| CN1300333C (en) * | 2003-04-17 | 2007-02-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation of gene chip for digagnosingantrax baiuus and its application |
| CN100402665C (en) * | 2005-11-30 | 2008-07-16 | 辽宁省农业科学院植物保护研究所 | Method for preparing grape anthracnose disease biochip |
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2009
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300333C (en) * | 2003-04-17 | 2007-02-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation of gene chip for digagnosingantrax baiuus and its application |
| CN100402665C (en) * | 2005-11-30 | 2008-07-16 | 辽宁省农业科学院植物保护研究所 | Method for preparing grape anthracnose disease biochip |
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| Luna VA et al.Bacillus sp. CBD119 plasmid putative CapB，putative CapC，and putative CapA genes，complete cds，GenBank No：DQ517348，3106 bp DNA linear.《NCBI》.2006,1-2. * |
| Makino S et al.B.anthracis encapsulation protein genes (capA, capB, and capC),complete cds,GenBank No:M24150,3244 bp DNA linear.《NCBI》.1993,1-2. * |
| regan jf et al.Environmental monitoring for biological threat agents using the autonomous pathogen detection system with multiplexed polymerase chain reaction.《anal chem》.2008,第80卷(第19期),7422-7429. * |
| Rolfs A et al.Synthetic construct Bacillus anthracis clone FLH231089.01L capB gene,complete sequence,GenBank No:EF036726,1395 bp DNA linear.《NCBI》.2006,1. * |
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