CN103981284B - A kind of method and test kit detecting foot and mouth disease virus in aerosol - Google Patents
A kind of method and test kit detecting foot and mouth disease virus in aerosol Download PDFInfo
- Publication number
- CN103981284B CN103981284B CN201410188272.9A CN201410188272A CN103981284B CN 103981284 B CN103981284 B CN 103981284B CN 201410188272 A CN201410188272 A CN 201410188272A CN 103981284 B CN103981284 B CN 103981284B
- Authority
- CN
- China
- Prior art keywords
- primer
- mouth disease
- foot
- type
- disease virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a kind of method detecting foot and mouth disease virus in aerosol: (1) gathers aerosol sample; (2) cDNA of virus in aerosol sample is extracted; (3) detect: carry out pcr amplification; (4) Criterion curve and solubility curve: the typical curve setting up positive criteria plasmid, and the solubility curve of amplification system; (5) whether judge in aerosol sample containing foot and mouth disease virus.The invention also discloses Auele Specific Primer (as SEQ? ID? shown in NO.1 ~ 8) and test kit (by Auele Specific Primer, RT reaction buffer, AMV ThermoScript II,
premix? Ex? Taq
tMiI, standard positive plasmid, to form without the water of RNase).In detection aerosol of the present invention, the method for foot and mouth disease virus and test kit both can be used for the aerocolloidal foot and mouth disease virus of plant and had detected and Serotypes, also can be used for the direct-detection of clinical sample, also can be used for the diagnosis of the non-diseases such as scientific research.
Description
Technical field
The present invention relates to a kind of method being detected foot and mouth disease virus in aerosol by fluorescent quantitation SYBRGreenI method somatotype, and Auele Specific Primer, test kit, belong to field of biological detection.
Background technology
Foot and mouth disease is that acute, hot, high degree in contact sexually transmitted disease occurs the artiodactyls such as pig, ox, sheep caused by foot and mouth disease virus, once break out, must slaughter the animal of infection and contact infection.Foot and mouth disease is classified as statutory report epidemic disease by OIE, be classified as first of a class animal epidemic by China, not only can cause huge direct economic loss, and the sound development of serious harm livestock industry and the foreign trade of related products, to the politics of country, economy, there is far-reaching influence.
Foot and mouth disease virus has the blood serum subtype of 7 serotypes and more than 65, without cross immunity phenomenon between serotype, is thus difficult to control.China is the popular country of foot and mouth disease, Major Epidemic O type, A type and Asia1 type foot and mouth disease.Within 2005, China's big area has broken out Asia1 type foot and mouth disease; within 2009 and 2013, broken out A type foot and mouth disease, in recent years, what all have an O type foot and mouth disease distributes with popular; particularly along with China's aquaculture is intensive, the fast development of mass-producing, the propagation risk of foot and mouth disease is also come larger.
Due to cultivated animals highly dense, infected animal is breathed out, is drained a large amount of pathogenic micro-organisms, causes in house that high concentration microorganism is aerocolloidal gathers, and by the exchange of the inside and outside gas of house, pathogenic micro-organism, along with atmospheric propagation, diffusion, causes environmental organism to pollute and infectious disease transmission.Foot and mouth disease virus can form aerosol in atmosphere, and virus can be disseminated to the place outside 50 ~ 100 kilometers with the wind, and velocity of propagation is fast, distance, is difficult to defence.Therefore, being applicable to the aerocolloidal detection method of foot and mouth disease virus by setting up one, supporting technology will being provided for foot and mouth disease forecasting and warning.
The detection method being applied to aerosol virus at present mainly contains culture method and molecular biology method, real-time fluorescence quantitative PCR determines that DNA in sample (or cDNA) copy number is the most responsive, molecular biology method the most accurately at present, there is sensitivity and specificity is high, level of automation is high, the feature such as pollution-free, real-time and accurate, can be applicable to cause of disease trace detection.But have no report about aerocolloidal foot and mouth disease virus fluorescence quantitative PCR detection and classifying method thereof.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of method being detected foot and mouth disease virus in aerosol by fluorescent quantitation SYBRGreenI method somatotype, and Auele Specific Primer, test kit.
The present invention is achieved by the following technical solutions:
Detect a method for foot and mouth disease virus in aerosol, step is as follows:
(1) aerosol sample is gathered;
Further, the method gathering aerosol sample is: adopt MS-1 type multifunctional microbial sampling thief, with the phosphate buffered saline buffer of 10mLpH value 7.0 (PBS) for sampling media, gather 30min according to the sampling flow of 12.5L/min, collect the aerosol sample in plant's environment;
(2) cDNA of virus in aerosol sample is extracted;
Further, the method extracting cDNA is: get 2mL aerosol sample, and application viral RNA extracts test kit (commercially produced product, conventional kit of the prior art) and extracts total serum IgE; Refer again to Reverse Transcription box (commercially produced product, conventional kit of the prior art) specification sheets and carry out reverse transcription, obtain cDNA;
(3) detect: with the cDNA of said extracted for template, adopt test kit to carry out SYBRGreen I real-time fluorescence quantitative PCR, specific as follows:
Described test kit by Auele Specific Primer, RT reaction buffer, AMV ThermoScript II,
premixExTaq
tMiI, standard positive plasmid, to form without the water of RNase.
Described standard positive plasmid comprises positive plasmid pEASY-T3-5 ' UTR, pEASY-T3-O-VP1, pEASY-T3-A-VP1 and pEASY-T3-A1-VP1; For the preparation of typical curve and the calculating of aerosol foot and mouth disease virus copy number.
Described positive plasmid pEASY-T3-5 ' UTR is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment of the 28-203 position Nucleotide of sequence shown in SEQIDNO.9, and method of attachment is affiliated field routine techniques.
Described positive plasmid pEASY-T3-O-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in IDNO.10, and method of attachment is affiliated field routine techniques.
Described positive plasmid pEASY-T3-A-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.11, and method of attachment is affiliated field routine techniques.
Described positive plasmid pEASY-T3-A1-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.12, and method of attachment is affiliated field routine techniques.
Described Auele Specific Primer is selected from universal primer that foot and mouth disease virus detects to or/and O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses are differentiated to detect primer pairs;
The sequence that the universal primer of described foot and mouth disease virus detection is right is as follows:
Primer 1-5 ' UTR-F:5 '-CGTTGCACTCCACACTTAC-3 ' (SEQIDNO.1);
Primer 2-5 ' UTR-R:5 '-GACCAAGTAGGCGGAAAG-3 ' (SEQIDNO.2);
O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses differentiate that the sequence detecting primer pair is as follows:
O type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 3-O-VP1F:5 '-GCAACTCAGGTCCAGAGGCGTC-3 ' (SEQIDNO.3);
Primer 4-O-VP1R:5 '-CAGTGTGTGAGCAGGGATCTGCATC-3 ' (SEQIDNO.4);
A type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 5-A-VP1F:5 '-AGACACAGGCTCAGCGACGTC-3 ' (SEQIDNO.5);
Primer 6-A-VP1R:5 '-GGCTGCACGCAAAAGGGCACCCACC-3 ' (SEQIDNO.6);
Asia1 type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 7-A1-VP1F:5 '-GAAACTCAGACAGCCAGGCGGC-3 ' (SEQIDNO.7);
Primer 8-A1-VP1R:5 '-CGCAGACCGCAGCAGGCTCCAACC-3 ' (SEQIDNO.8).
Amplification reaction system is: the amplification system of 20 μ L, comprises 2 × SYBRPremixExTaqII10 μ L, the upstream primer of 10 μm of ol/L and each 0.5 μ L of downstream primer, positive plasmid 1 μ L, RNaseFreeddH
2o supplies 20 μ L.
Amplification condition is: denaturation 95 DEG C of 30s, then extends 30s according to 95 DEG C of sex change 5s, 65 DEG C of annealing, carries out 40 circulations; Solubility curve is 95 DEG C of 5s, 65 DEG C of 60s, 95 DEG C of Continuous; Last 50 DEG C of 30s terminate reaction.
(4) Criterion curve and solubility curve: the typical curve setting up positive criteria plasmid, and the solubility curve of amplification system;
(5) judge: when Auele Specific Primer used is the universal primer pair of foot and mouth disease virus detection, if solubility curve is S type curve, then show in aerosol sample containing foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing foot and mouth disease virus;
When Auele Specific Primer used is O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses discriminating detection primer pairs, if correspond to O type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show to contain O type foot and mouth disease virus in aerosol sample; If solubility curve is straight line, then show in aerosol sample not containing O type foot and mouth disease virus;
If correspond to A type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show in aerosol sample containing A type foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing A type foot and mouth disease virus;
If correspond to Asia1 type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show in aerosol sample containing Asia1 type foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing Asia1 type foot and mouth disease virus.
In detection aerosol of the present invention, the method for foot and mouth disease virus and test kit both can be used for the aerocolloidal foot and mouth disease virus of plant and had detected and Serotypes, also can be used for the direct-detection of clinical sample, also can be used for the diagnosis of the non-diseases such as scientific research.
Accompanying drawing explanation
The typical curve of Fig. 1: foot and mouth disease virus SYBRGreenI real-time quantitative PCR, the copy number that in figure, each point is corresponding is: A:3.0 × 10
0copy; B:3.0 × 10
1copy; C:3.0 × 10
2copy; D:3.0 × 10
3copy; E5:3.0 × 10
4copy; F:3.0 × 10
5copy.
The solubility curve of Fig. 2: foot and mouth disease virus SYBRGreenI real-time quantitative PCR, wherein, negative control is with equivalent empty carrier pEASY-T3 for template, and other templates are that standard positive plasmid is respectively 3.0 × 10
5-3.0 × 10
0copy.
The specificity curve of Fig. 3: foot and mouth disease virus SYBRGreenI real-time quantitative PCR, wherein, 1:3.0 × 10
4the standard positive plasmid of copy; 2:O type foot and mouth disease virus; 3:A type foot and mouth disease virus; 4:Asia1 type foot and mouth disease virus; 5: infectious bovine rhinotrachetis virus; 6: bovine enteroviruses; 7: ox stream fever virus; 8: bovine viral diarrhea virus; 9: vesicular stomatitis virus; 10: Pestivirus suis; 11: herpesvirus suis; 12:ddH
2o.
The sensitivity technique of Fig. 4: foot and mouth disease virus SYBRGreenI real-time quantitative PCR, wherein, 1:3.0 × 10
6copy; 2:3.0 × 10
5copy; 3:3.0 × 10
4copy; 4:3.0 × 10
3copy; 5:3.0 × 10
2copy; 6:3.0 × 10
1copy; 7:3.0 × 10
0copy; 8:1.0 × 10
0copy; 9: negative control (with equivalent pEASY-T3 for template).
The SYBRGreenI real-time quantitative PCR solubility curve of Fig. 5: O type foot and mouth disease virus.
The SYBRGreenI real-time quantitative PCR solubility curve of Fig. 6: A type foot and mouth disease virus.
The SYBRGreenI real-time quantitative PCR solubility curve of Fig. 7: Asia1 type foot and mouth disease virus.
Fig. 8: the detection curve of aerosol sample foot and mouth disease virus, wherein, A and B detects positive sample.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Reverse Transcription box (CodeNo.:D2639A),
premixExTaq
tMiI test kit (CodeNo.:RR820A), purchased from precious biotechnology (Dalian) company limited.Universal detector primer (primer 1 and primer 2); O type, A type, Asia1 type 3 kinds of Serotypes primers (primer 3 and primer 4, primer 5 and primer 6, primer 7 and primer 8), primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
The preparation of embodiment 1 standard substance positive plasmid
1. the Design and synthesis of primer
By carrying out sequence alignment to foot and mouth disease virus in GeneBank 5 ' UTR gene, determining conservative region, designing 1 pair of universal primer, for SEQIDNO.1 and SEQIDNO.2 in sequence table, can detect foot and mouth disease virus 7 serotypes, amplify PCR fragment, for the structure of positive criteria plasmid; Then according to the difference of FMDV VP1 sequence, 3 pairs of primers are designed respectively, can to O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease virus somatotypes.O type foot and mouth disease virus serotype specific primer is SEQIDNO.3 and SEQIDNO.4 in sequence table; A type foot and mouth disease virus serotype specific primer is sequence SEQIDNO.5 in sequence table and SEQIDNO.6; Asia1 type foot and mouth disease virus serotype specific primer is respectively SEQIDNO.7 and SEQIDNO.8 in sequence table.The PCR fragment that 3 kinds of Serotypes primers increase, for the structure of positive criteria plasmid.All primers are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
2. the preparation of foot and mouth disease virus cDNA
Extract test kit specification sheets with reference to viral DNA/RNA, extract O type, A type, Asia1 type foot and mouth disease virus RNA respectively, carry out reverse transcription with reference to Reverse Transcription box specification sheets, obtain cDNA.
3. the preparation of standard substance
The cDNA prepared with aforesaid method is template, carries out pcr amplification, and reaction system is 50 μ L:10 × PCRBufferII (Mg
2+plus) 5 μ L, 2.5mMdNTPMixture8 μ L, rTaq0.5 μ L (5U/ μ L), cDNA template 2 μ L, add each 2 μ L (primer 1 and the primer 2s of upstream and downstream primer pair of 10 μm of ol/L respectively, primer 3 and primer 4, primer 5 and primer 6, primer 7 and primer 8), sterilizing ultrapure water adds to 50 μ L.Response procedures is 94 DEG C of denaturation 3min, then 94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 20s, 35 circulations; 72 DEG C extend 5min, 4 DEG C of preservations.
PCR primer is through 1% agarose gel electrophoresis, reclaim object segment, be cloned into pEAST-T3 carrier, recombinant plasmid serves Hai Shenggong order-checking, sequence shown in SEQIDNO.9 in sequencing result and sequence table, 10,11,12 is compared, correct recombinant plasmid is positive, is distinguished called after pEASY-T3-5 ' UTR, pEASY-T3-O-VP1, pEASY-T3-A-VP1, pEASY-T3-A1-VP1.
Using pEASY-T3-5 ' UTR plasmid as positive criteria product, with nucleic acid-protein analyser (NanoPhotometer
tMp300) measure plasmid concentration, calculate the DNA copy number in every μ L plasmid according to the following equation, result copy number is 3.2 × 10
10copy/μ L, using this plasmid as standard substance.
Copy number (copy/μ L)=concentration (g/ μ L) ÷ molecular weight (g/mol) × 6 × 10
23
The SYBR Green I fluorescent quantitation RT-PCR reaction system of embodiment 2 foot and mouth disease virus universal primer and the optimization of condition
The optimization of 1.AMV ThermoScript II consumption
Through using the test-results of different concns AMV ThermoScript II to compare, selected 5U is as the optimum amount of AMV in reaction system.
2. the optimization of primer concentration
In reaction system, primer concentration is done multiple proportions serial dilution from 0.1 μm of ol/L to 1.6 μm of ol/L respectively, mutually after combination through quantitative real time PCR Instrument (
480II) detect, by the com-parison and analysis of test-results, the best primer final concentration 0.3 μm of ol/L determined.And the reaction system of foot and mouth disease virus somatotype composite S YBRGreenI fluorescence quantitative RT-RCR, the best primer final concentration of primer 3, primer 4, primer 7 and primer 8 0.2 μm of ol/L, primer 5 and primer 6 optimal final concentration are 0.3 μm of ol/L.
3. the optimization of reaction conditions
Carry out annealing temperature from the optimization of 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C, 66 DEG C, 67 DEG C, all primers are when 65 DEG C of annealing temperatures, and fluorescent signal is best.
The foundation of the SYBR Green I fluorescent quantitation RT-PCR detection method of embodiment 3 foot and mouth disease virus universal primer
1. the determination of typical curve and solubility curve analysis
The determination of quantitative fluorescent PCR typical curve, carries out quantitatively, then being undertaken being diluted to 3 × 10 by 10 times to plasmid pEAST-T3-5 ' UTR
0-3 × 10
9copy number/μ L, carries out the amplification of SYBRGreenI real-time fluorescence quantitative PCR using the copy number of dilution as template, Criterion curve.Amplification reaction system is 20 μ L:2 × SYBRPremixExTaqII10 μ L, and concentration is 5 ' UTR-F and 5 ' UTR-R each 0.5 μ L, positive plasmid pEAST-T3-5 ' UTR1 μ L of 10 μm of ol/L, RNaseFreeddH
2o supplies 20 μ L.Negative control (replacing template with equivalent pEAST-T3) is set simultaneously.Then carry out the amplification of typical curve, reaction conditions is denaturation 95 DEG C of 30s, then extends 30s carry out 40 circulations according to 95 DEG C of sex change 5s, 65 DEG C of annealing; Solubility curve is 95 DEG C of 5s, 65 DEG C of 60s, 95 DEG C of Continuous; Last 50 DEG C of 30s terminate reaction.
As shown in Figure 1, each point in a straight line, namely shows that typical curve is good to typical curve.
As shown in Figure 2, solubility curve peak is single for solubility curve, shows that specific amplification goes out positive peak, does not have primer dimer or other non-positive peak to occur.
The specific test of 2.SYBRGreenI real-time fluorescence quantitative PCR
Extract test kit specification sheets with reference to viral DNA/RNA, extract the DNA of infectious bovine rhinotrachetis virus and herpesvirus suis; Extract bovine enteroviruses, ox stream fever virus, bovine viral diarrhea virus, vesicular stomatitis virus, Pestivirus suis RNA, carry out reverse transcription with reference to Reverse Transcription box specification sheets, obtain cDNA.
The SYBRGreenI real time fluorescence quantifying PCR method set up according to above-mentioned steps carries out specific test, respectively with the DNA of foot and mouth disease virus cDNA, infectious bovine rhinotrachetis virus DNA, bovine enteroviruses cDNA, ox stream fever virus cDNA, bovine viral diarrhea virus cDNA, vesicular stomatitis virus cDNA, swine fever virus cDNA, herpesvirus suis for template, with ddH
2o is negative control, carries out SYBRGreenI real-time fluorescence quantitative PCR.
The specific result of foot and mouth disease virus is detected under 530nm exciting light, as shown in Figure 3,1:O type foot and mouth disease virus; 2:A type foot and mouth disease virus; 3:Asia1 type foot and mouth disease virus; 4: infectious bovine rhinotrachetis virus; 5: bovine enteroviruses; 6: ox stream fever virus; 7: bovine viral diarrhea virus; 8: vesicular stomatitis virus; 9: Pestivirus suis; 10: herpesvirus suis; 11:3.0 × 10
4copy number/μ L standard positive plasmid; 12:ddH
2o.As can be seen from the figure, 1-4 has the specificity fluorescent curve of corresponding bacterium, and 5-12 does not all have specificity fluorescent curve, and the primer amplification foot and mouth disease virus specificity designed by confirmation is good, detects viral no cross reaction with other.Therefore, the universal primer 5 ' UTR-F of step 1 foot and mouth disease virus used, 5 ' UTR-R, the SYBRGreenI real-time fluorescence quantitative PCR detection method of foundation can be used for qualification unknown sample and whether there is foot and mouth disease virus nucleic acid.
The sensitivity replica test of 3.SYBRGreenI real-time fluorescence quantitative PCR
With pEASY-T3-5 ' UTR plasmid prepared by 10 times of serial dilution above-mentioned steps one, obtaining copy number is 1 × 10
5-1 × 10
0the serial dilutions of copy/μ L, using the diluent containing different plasmid copy numbers as template, arrange an equivalent pEASY-T3 plasmid is negative control simultaneously, carries out SYBR Green I fluorescent quantitation pcr amplification.Wherein, the reaction system after SYBR Green I fluorescent quantitation PCR adopts embodiment 2 to optimize and reaction conditions.
SYBR Green I fluorescent quantitation pcr amplification result is detected under 530nm exciting light, as shown in Figure 4,1:3.0 × 10
6copy number/μ L; 2:3.0 × 10
5copy number/μ L; 3:3.0 × 10
4copy number/μ L; 4:3.0 × 10
3copy number/μ L; 5:3.0 × 10
2copy number/μ L; 6:3.0 × 10
1copy number/μ L; 7:3.0 × 10
0copy number/μ L; 8:1.0 × 10
0copy number/μ L; 9: negative control (with equivalent pEASY-T3 for template).From the fluorescence curve figure, fluorescence curve is still had to monitoring 3 copies of foot and mouth disease virus, show that SYBR Green I fluorescent quantitation PCR detection method of the present invention is 3 copies to the sensitivity of foot and mouth disease virus, far above conventional PCR method detection sensitivity.
The replica test of 4.SYBRGreenI real-time fluorescence quantitative PCR
According to the SYBR Green I fluorescent quantitation PCR method set up in above-mentioned steps 1, choose 3.0 × 10
4copy number/μ L and 3.0 × 10
2copy number/μ L two different concns pEASY-T3-5 ' UTR plasmid is template, simultaneously with equivalent pEASY-T3 for negative control, each template concentrations does three repetitions, is verified batch interior repeatability of quantitative fluorescent PCR by the standard deviation (S) and the variation coefficient (CV) calculating Ct value; PEASY-T3-5 ' the UTR plasmid DNA that alternative gets a concentration is as template, and carry out fluorescent PCR amplification, interval 7d repeats once, carries out 3 times altogether and repeats, then calculate the variation coefficient (CV) of Ct value, checking quantitative fluorescent PCR batch between repeated.
Choose 3.0 × 10
4copy number/μ L and 3.0 × 10
2pEASY-T3-5 ' the UTR plasmid of copy number/μ L bis-concentration makes revision test, and the Ct value of its gained is respectively 16.25,16.78 and 18.92,18.47, and its Ct value variation coefficient is all less than 5%; Choose 1.0 × 10
4pEASY-T3-5 ' the UTR plasmid of copy number/μ L, carried out fluorescent PCR mensuration totally three times at interval of 7 days, the variation coefficient of its Ct value is also less than 5%.Result shows, the SYBR Green I fluorescent quantitation PCR method of foundation is reproducible.
The foundation of embodiment 4 foot and mouth disease virus somatotype SYBR Green I fluorescent quantitation RT-PCR detection method
Positive plasmid pEAST-T3-O-VP1, pEAST-T3-A-VP1, pEAST-T3-A1-VP1 are diluted, gets 1 × 10
2the diluent of copy number/μ L is template, carries out the somatotype detection of O type, A type, Asia1 type foot and mouth disease virus respectively.The amplification reaction system that single somatotype detects is 20 μ L:2 × SYBRPremixExTaqII10 μ L, 10 μm of each 0.3 μ L of each primer pair of ol/L, the positive plasmid 1 μ L corresponding with primer, RNaseFreeddH
2o supplies 20 μ L, with equivalent pEAST-T3 for negative control.Solubility curve is 95 DEG C of 5s, 65 DEG C of 60s, 95 DEG C of Continuous; Last 50 DEG C of 30s terminate reaction.
The solubility curve of O type, A type, Asia1 type foot and mouth disease virus somatotype is as shown in Fig. 5 ~ Fig. 7, and solubility curve peak is single, does not have primer dimer or other non-positive peak to occur.Through the Tm value of each serotype of computed in software, the solubility curve Tm value of O type, A type, Asia1 type foot and mouth disease virus is respectively: 88.53+0.02,88.53+0.03,84.21+0.04.
The detection of embodiment 5 aerosol sample
1. aerosol sample collection
Select the different zones (cow house, playground, milking parlour etc.) of diary farm, application MS-I type air microbe sampling box, by the Porton samplers sample air sample of multifunctional microbial sampling thief (Qingdao, MS-1 type).First install trivet, sampling thief placing height is apart from ground 1.5m, then 10mL sterile phosphate buffer (PBS) is injected Proton sampling thief, drips a sweet oil simultaneously, puts into tripod fixed support internal fixtion.Receive on main frame air intake by an end of Porton impact type current stabilizer, the other end rubber hose is connected on the air outlet of Proton sampling thief.Sampling flow is 5 ~ 35L/min, and the sampling time is 30min.After sampling terminates, the collection liquid in sampling thief is collected the preservation of 15mL centrifuge tube to be used for detecting.
2. the preparation of template
Aerosol absorption liquid, gets about 1mL, adopts viral RNA to extract test kit, extracts RNA.Reverse transcription obtains cDNA, and-20 DEG C of storages are for subsequent use.
3. the detection of clinical aerosol sample
Sample to be tested is the aerosol sample that dairy cow farm different location, different areas, Shandong Province is collected, and amounts to 36 parts, extracts the geneome RNA of these aerosol samples respectively, reverse transcription cDNA.
Be respectively template with the cDNA of the 36 parts of aerosol samples extracted, the SYBR Green I fluorescent quantitation PCR method set up according to embodiment 3 detects.If the reaction result under 530nm exciting light is S type curve, then contain foot and mouth disease virus in sample to be tested; If reaction result is straight line, in sample to be tested, there is no foot and mouth disease virus.
After measured, under 530nm exciting light, detect in sample and occur that the sample of S type curve has 2 parts, for A and B, detecting sample is having 34 parts (see Fig. 8) of straight line, have 2 parts in interpret sample for the foot and mouth disease virus positive, 34 increment product are that foot and mouth disease virus is negative, namely not containing foot and mouth disease virus.
Contriver adopts for foot and mouth disease 5 ' UTR gene (SEQIDNO.9) after above 36 parts of aerosol samples being extracted geneome RNA respectively simultaneously) method of carrying out sequencing confirms the exactness of SYBR Green I fluorescent quantitation PCR detection method provided by the present invention further.Sequencing result shows, and 2 increment product are that foot and mouth disease virus is positive, and all the other 34 increment product are that foot and mouth disease virus is negative, sequencing result and SYBR Green I fluorescent quantitation PCR detection method result completely the same.
Claims (6)
1. a method for foot and mouth disease virus in the detection aerosol of non-diagnostic object, is characterized in that: step is as follows:
(1) aerosol sample is gathered;
(2) cDNA of virus in aerosol sample is extracted;
(3) detect: with the cDNA of said extracted for template, adopt test kit to carry out SYBRGreen I real-time fluorescence quantitative PCR, specific as follows:
Described test kit is by Auele Specific Primer, RT reaction buffer, AMV ThermoScript II, SYBRPremixExTaq
tMiI, standard positive plasmid, to form without the water of RNase;
Described standard positive plasmid comprises positive plasmid pEASY-T3-5 ' UTR, pEASY-T3-O-VP1, pEASY-T3-A-VP1 and pEASY-T3-A1-VP1;
Described positive plasmid pEASY-T3-5 ' UTR is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment of the 28-203 position Nucleotide of sequence shown in SEQIDNO.9;
Described positive plasmid pEASY-T3-O-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in IDNO.10;
Described positive plasmid pEASY-T3-A-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.11;
Described positive plasmid pEASY-T3-A1-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.12;
Described Auele Specific Primer is that the universal primer that foot and mouth disease virus detects is differentiated to detect primer pair to O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses;
The sequence that the universal primer of described foot and mouth disease virus detection is right is as follows:
Primer 1-5 ' UTR-F:5 '-CGTTGCACTCCACACTTAC-3 ';
Primer 2-5 ' UTR-R:5 '-GACCAAGTAGGCGGAAAG-3 ';
O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses differentiate that the sequence detecting primer pair is as follows:
O type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 3-O-VP1F:5 '-GCAACTCAGGTCCAGAGGCGTC-3 ';
Primer 4-O-VP1R:5 '-CAGTGTGTGAGCAGGGATCTGCATC-3 ';
A type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 5-A-VP1F:5 '-AGACACAGGCTCAGCGACGTC-3 ';
Primer 6-A-VP1R:5 '-GGCTGCACGCAAAAGGGCACCCACC-3 ';
Asia1 type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 7-A1-VP1F:5 '-GAAACTCAGACAGCCAGGCGGC-3 ';
Primer 8-A1-VP1R:5 '-CGCAGACCGCAGCAGGCTCCAACC-3 ';
(4) Criterion curve and solubility curve: the typical curve setting up positive criteria plasmid, and the solubility curve of amplification system;
(5) judge: when Auele Specific Primer used is the universal primer pair of foot and mouth disease virus detection, if solubility curve is S type curve, then show in aerosol sample containing foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing foot and mouth disease virus;
When Auele Specific Primer used is O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses discriminating detection primer pairs, if correspond to O type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show to contain O type foot and mouth disease virus in aerosol sample; If solubility curve is straight line, then show in aerosol sample not containing O type foot and mouth disease virus;
If correspond to A type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show in aerosol sample containing A type foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing A type foot and mouth disease virus;
If correspond to Asia1 type foot and mouth disease virus to differentiate that the solubility curve detecting primer pair is S type, then show in aerosol sample containing Asia1 type foot and mouth disease virus; If solubility curve is straight line, then show in aerosol sample not containing Asia1 type foot and mouth disease virus;
In described step (3), the amplification reaction system of PCR is: the amplification system of 20 μ L, comprises 2 × SYBRPremixExTaqII10 μ L, the upstream primer of 10 μm of ol/L and each 0.5 μ L of downstream primer, positive plasmid 1 μ L, RNaseFreeddH
2o supplies 20 μ L;
In described step (3), the amplification condition of PCR is: denaturation 95 DEG C of 30s, then extends 30s according to 95 DEG C of sex change 5s, 65 DEG C of annealing, carries out 40 circulations; Solubility curve is 95 DEG C of 5s, 65 DEG C of 60s, 95 DEG C of continuation; Last 50 DEG C of 30s terminate reaction.
2. the method for foot and mouth disease virus in detection aerosol according to claim 1, it is characterized in that: in described step (1), the method gathering aerosol sample is: adopt MS-1 type multifunctional microbial sampling thief, with the phosphate buffered saline buffer of 10mLpH value 7.0 for sampling media, gather 30min according to the sampling flow of 12.5L/min, collect aerosol sample.
3. the method for foot and mouth disease virus in detection aerosol according to claim 1, is characterized in that: in described step (2), and the method extracting cDNA is: get 2mL aerosol sample, and application viral RNA extracts test kit and extracts total serum IgE; Apply Reverse Transcription box again and carry out reverse transcription, obtain cDNA.
4. for detecting the Auele Specific Primer of foot and mouth disease virus, it is characterized in that: described Auele Specific Primer is that the universal primer that foot and mouth disease virus detects is differentiated to detect primer pair to O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses;
The sequence that the universal primer of described foot and mouth disease virus detection is right is as follows:
Primer 1-5 ' UTR-F:5 '-CGTTGCACTCCACACTTAC-3 ';
Primer 2-5 ' UTR-R:5 '-GACCAAGTAGGCGGAAAG-3 ';
O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses differentiate that the sequence detecting primer pair is as follows:
O type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 3-O-VP1F:5 '-GCAACTCAGGTCCAGAGGCGTC-3 ';
Primer 4-O-VP1R:5 '-CAGTGTGTGAGCAGGGATCTGCATC-3 ';
A type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 5-A-VP1F:5 '-AGACACAGGCTCAGCGACGTC-3 ';
Primer 6-A-VP1R:5 '-GGCTGCACGCAAAAGGGCACCCACC-3 ';
Asia1 type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 7-A1-VP1F:5 '-GAAACTCAGACAGCCAGGCGGC-3 ';
Primer 8-A1-VP1R:5 '-CGCAGACCGCAGCAGGCTCCAACC-3 '.
5. Auele Specific Primer according to claim 4 detects the application in the test kit of foot and mouth disease virus in preparation.
6. for detecting a test kit for foot and mouth disease virus, it is characterized in that: described test kit is by Auele Specific Primer, RT reaction buffer, AMV ThermoScript II, SYBRPremixExTaq
tMiI, standard positive plasmid, to form without the water of RNase;
Described standard positive plasmid comprises positive plasmid pEASY-T3-5 ' UTR, pEASY-T3-O-VP1, pEASY-T3-A-VP1 and pEASY-T3-A1-VP1;
Described positive plasmid pEASY-T3-5 ' UTR is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment of the 28-203 position Nucleotide of sequence shown in SEQIDNO.9;
Described positive plasmid pEASY-T3-O-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in IDNO.10;
Described positive plasmid pEASY-T3-A-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.11;
Described positive plasmid pEASY-T3-A1-VP1 is the recombinant plasmid obtained after being connected with pEASY-T3 carrier by the sequence fragment shown in SEQIDNO.12;
Described Auele Specific Primer is that the universal primer that foot and mouth disease virus detects is differentiated to detect primer pair to O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses;
The sequence that the universal primer of described foot and mouth disease virus detection is right is as follows:
Primer 1-5 ' UTR-F:5 '-CGTTGCACTCCACACTTAC-3 ';
Primer 2-5 ' UTR-R:5 '-GACCAAGTAGGCGGAAAG-3 ';
O type, A type, Asia1 type 3 kinds of serotype foot and mouth disease viruses differentiate that the sequence detecting primer pair is as follows:
O type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 3-O-VP1F:5 '-GCAACTCAGGTCCAGAGGCGTC-3 ';
Primer 4-O-VP1R:5 '-CAGTGTGTGAGCAGGGATCTGCATC-3 ';
A type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 5-A-VP1F:5 '-AGACACAGGCTCAGCGACGTC-3 ';
Primer 6-A-VP1R:5 '-GGCTGCACGCAAAAGGGCACCCACC-3 ';
Asia1 type foot and mouth disease virus differentiates that the sequence detecting primer pair is as follows:
Primer 7-A1-VP1F:5 '-GAAACTCAGACAGCCAGGCGGC-3 ';
Primer 8-A1-VP1R:5 '-CGCAGACCGCAGCAGGCTCCAACC-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410188272.9A CN103981284B (en) | 2014-05-06 | 2014-05-06 | A kind of method and test kit detecting foot and mouth disease virus in aerosol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410188272.9A CN103981284B (en) | 2014-05-06 | 2014-05-06 | A kind of method and test kit detecting foot and mouth disease virus in aerosol |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103981284A CN103981284A (en) | 2014-08-13 |
| CN103981284B true CN103981284B (en) | 2016-03-09 |
Family
ID=51273455
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410188272.9A Active CN103981284B (en) | 2014-05-06 | 2014-05-06 | A kind of method and test kit detecting foot and mouth disease virus in aerosol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103981284B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946795B (en) * | 2015-06-01 | 2018-03-20 | 山东省农业科学院奶牛研究中心 | Primer, probe and kit for Site Detection various serotype foot and mouth disease virus |
| CN104862420B (en) * | 2015-06-01 | 2017-10-13 | 山东省农业科学院奶牛研究中心 | RPA Sidestream chromatographies detect primer, probe and the kit of foot and mouth disease virus aerosol |
| CN107345258A (en) * | 2017-08-28 | 2017-11-14 | 黑龙江珍宝岛药业股份有限公司 | A kind of primer combination for detecting virus and application, kit |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
| CN102329879A (en) * | 2011-10-14 | 2012-01-25 | 山东农业大学 | Rapid identification method of Brucellosis aerosol by using fluorescent quantitative PCR (polymerase chain reaction) |
-
2014
- 2014-05-06 CN CN201410188272.9A patent/CN103981284B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
| CN102329879A (en) * | 2011-10-14 | 2012-01-25 | 山东农业大学 | Rapid identification method of Brucellosis aerosol by using fluorescent quantitative PCR (polymerase chain reaction) |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103981284A (en) | 2014-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103981261B (en) | One detects and brucellar method in differential diagnosis aerosol | |
| CN104862420B (en) | RPA Sidestream chromatographies detect primer, probe and the kit of foot and mouth disease virus aerosol | |
| CN103509877B (en) | A kind of PCR kit for fluorescence quantitative for detecting PRV and application thereof | |
| CN102140543B (en) | Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases | |
| CN103981284B (en) | A kind of method and test kit detecting foot and mouth disease virus in aerosol | |
| CN104032034A (en) | Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit | |
| CN103981285B (en) | A kind of detect the method for bovine viral diarrhea virus in aerosol | |
| CN103981283B (en) | A kind of method detecting infectious bovine rhinotrachetis virus in aerosol | |
| CN102453762B (en) | Kit and method for detecting porcine proliferative enteropathy (PPE) pathogenic bacteria Lawsonia Intracellularis | |
| CN102329879B (en) | Rapid identification method of Brucellosis aerosol by using fluorescent quantitative PCR (polymerase chain reaction) | |
| CN107988340A (en) | A kind of PCR amplification primer of quick detection mycoplasma ovine pneumoniae and its application | |
| CN104388594B (en) | A kind of Taqman Real-time PCR kit for detecting PRV (Pseudorabies virus) | |
| CN103725793A (en) | Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof | |
| CN103725794A (en) | Fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) primers, probes and method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) | |
| CN113186312B (en) | Molecular marker for distinguishing Brucella A19 vaccine strain and wild strain | |
| CN108624713A (en) | A kind of porcine pseudorabies vaccine virus differentiates the method and kit of detection with wild poison | |
| CN107686865A (en) | Detect primer pair, probe, kit and the method for I type bovine herpes virus | |
| CN108411014A (en) | Differentiate the primer and probe and detection method of A types and the dual real-time fluorescence quantitative PCR of Type B ox pasteurella multocida | |
| CN102329890B (en) | Primer, probe and method for detecting mouse adenovirus | |
| CN106435007A (en) | Primer, probe, kit and method for detecting bacillus erysipelatos-suis by fluorescent quantitative PCR (Polymerase Chain Reaction) | |
| CN105385683A (en) | Multiplex RT-PCR detection kit for porcine epidemic diarrhea viruses | |
| CN104946753A (en) | Specificity primer pair for cow mycoplasma detection, detection kit, as well as using method and application of detection kit | |
| CN109234432A (en) | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection soybean samping off | |
| CN104388593A (en) | Taqman Real-time PCR (polymerase chain reaction) kit for detecting porcine circovirus | |
| CN104372110A (en) | Reagent kit used for testing Taqman Real-time RT-PCR (reverse transcription-polymerase chain reaction) of peste des petits ruminants virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |