CN104404170A - General A-type double real-time fluorescence quantitation PCR detection primer and probe for foot and mouth disease viruses - Google Patents
General A-type double real-time fluorescence quantitation PCR detection primer and probe for foot and mouth disease viruses Download PDFInfo
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Abstract
The invention provides a general A-type double real-time fluorescence quantitation PCR (Polymerase Chain Reaction) detection primer and probe for foot and mouth disease viruses. The primer comprises P0, P1, P2, P3 and P4, and the probe comprises P5 and P6. Double real-time fluorescence quantitation PCR detection established based on the primer and the probe can realize quick identification and detection for three subtype general foot and mouth disease viruses, namely the foot and mouth disease viruses O, A, AsinaI in one reaction at the same time, and the detection can be completed in 1-2 h; the detection has the advantages of being quick, specific, sensitive, high in flux and the like, and the requirements for quickly identifying and detecting the foot and mouth disease viruses and A-type foot and mouth disease viruses in a large scale can be met.
Description
Technical field
The invention belongs to animal epidemic quarantine detection technique field, specifically, relate to that foot and mouth disease virus is universal, the double real-time fluorescence quantitative PCR of A type detects primer and probe.
Background technology
Foot and mouth disease (FMD) is by foot and mouth disease virus (Footand Mouth Disease Virus, the one that the Some Livestocks such as the pig FMDV) caused, ox, sheep and other domestic, wild artiodactyl suffer from altogether is acute, pernicious, high degree in contact sexually transmitted disease, and susceptible animal reaches kind more than 70.This sick route of transmission is many, speed is fast, once repeatedly worldwide outbreak of epidemic, causes huge politics, financial loss.OIE (OIE) is classified as first of category-A transmissible disease.At present, have the popular FMD of OIE member states of 2/3rds, the moment threatens safety of livestock without FMD countries and regions and Livestock Product Trade.Foot and mouth disease comprises 7 serotypes, China popular and threaten by neighbouring country mainly contain O, A, Asia I 3 serotypes, and cross-protection between each serotype is very poor.Therefore, utilize simply, the inspection method differential diagnosis of as early as possible foot and mouth disease being made to type is fast and accurately most important to control foot and mouth disease epidemic situation.Real-time fluorescence quantitative PCR (fluorescent quantitative PCR, referred to as FQ-PCR) is a kind of new diagnostic instrument, has sensitivity, fast and accurately feature, and realize the quantitative assay of template while nucleic acid rapid amplifying.The multi-joint real-time fluorescence PCR technology grown up at present can in same PCR reaction, and distinguish different nucleic acid fragments by the analysis of Tm value, this provides technical foundation for detecting multiple cause of disease simultaneously.
Summary of the invention
The object of this invention is to provide that a kind of can to detect O, A, Asia I 3 serotype foot and mouth disease viruses quickly and accurately universal and differentiate that the foot and mouth disease virus of A type foot and mouth disease virus is universal, the double real-time fluorescence quantitative PCR of A type detects primer and probe.
Another object of the present invention is to provide above-mentioned primer and probe is detecting foot and mouth disease virus and differentiating the application in A type foot and mouth disease virus.
In order to realize the object of the invention, foot and mouth disease virus provided by the invention is general, the double real-time fluorescence quantitative PCR of A type (double FQ-PCR) detects primer and probe, and described primer comprises:
P0:5 '-CGGGAAACGCATGAGCAGTA-3 ' (shown in SEQ ID NO:3 sequence);
P1:5 '-CGGCCTCTCATCCAACAGA-3 ' (shown in SEQ ID NO:4 sequence);
P2:5 '-ATTTTCCTTCAGGCGCTTGA-3 ' (shown in SEQ ID NO:5 sequence);
P3:5 '-AACCCCACCGCCTACCA-3 ' (shown in SEQ ID NO:6 sequence);
P4:5 '-GGTGTAAGGGAGCGCAAGTC-3 ' (shown in SEQ ID NO:7 sequence);
Described probe comprises:
Sequence 5' end shown in Probe-P5:5'-F1-TCATTTGTGAAACGCG-Q1-3'(SEQ ID NO:8 connects F1,3' end and connects Q1);
Sequence 5' end shown in Probe-P6:5'-F2-AAGCAGCCGTTTACG-Q2-3'(SEQ ID NO:9 connects F2,3' end and connects Q2);
Wherein, F1, F2 are fluorophor, and Q1, Q2 are non-fluorescence quenching group, and F1 is FAM, F2 be VIC, Q1 and Q2 is MGB.
Wherein, P0 is reverse transcription primer, obtains cDNA for carrying out reverse transcription to the total serum IgE of viral sample to be measured; P1, P2, P3, P4 and Probe-P5 and Probe-P6 are real-time fluorescence quantitative PCR primer and probe.
The present invention also provides the test kit detecting the universal and A type foot and mouth disease virus of foot and mouth disease virus for real-time fluorescence quantitative PCR containing above-mentioned primer and probe.
Wherein, can also contain positive reference substance and negative controls in test kit provided by the present invention, positive reference substance can be O type, A type and Asia I type foot and mouth disease virus type strain, and negative control can be water or other Virus Standard strain.
The present invention further provides the application in real-time fluorescence quantitative PCR detection foot and mouth disease virus and A type foot and mouth disease virus of above-mentioned primer and probe or test kit.Described application comprises the following steps:
1) extract the total serum IgE of viral sample to be measured, carry out reverse transcription and obtain cDNA; Wherein, reverse transcription system is: 5 × M-MLV damping fluid 4 μ L, 2.5mmol/L dNTP 4 μ L, primer P01 μ L, M-MLV ThermoScript II 0.5 μ L, RNase inhibitor 0.5 μ L, RNA 10 μ L; Reverse transcription condition is: 37 DEG C, 1h;
2) being cloned into by cDNA in pGEM-T Easy carrier, screening positive recombinant plasmid, take plasmid as template, carries out pcr amplification reaction;
3) PCR primer is analyzed.
Wherein, the reaction system of carrying out pcr amplification reaction use is counted with 25 μ l: P1, P2, P3, P4 final concentration is respectively 0.4 μm of ol/L, Probe-P5 final concentration is 0.6 μm of ol/L, Probe-P6 final concentration is 0.7 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, 10 × Ex Taq enzyme damping fluid 2.5 μ L, 5U/ μ L Ex Taq DNA enzymatic 0.25 μ L, 1.0 × 10
5the plasmid template 2 μ L of copy/μ L, supplies deionized water to final volume 25 μ L.
Pcr amplification reaction condition is: 94 DEG C 5 minutes; 94 DEG C 15 seconds, 60 DEG C 30 seconds, totally 40 circulations.
In this reaction, positive control and negative control need be done simultaneously, universal detection system is set respectively and A type detection system is FAM and VIC fluorescence channel.
In the present invention, the decision procedure of detected result can be: threshold setting is as the criterion with the vertex of threshold line just above normal negative controls amplification curve, and different instrument can adjust according to instrument noise situation.The detected result of negative control should without specific amplification curve, and Ct value > 30.0 or nothing, and the Ct value of positive control should≤27.0, and occur specific amplification curve, judge that detected result is set up.Be discontented with as negative control and positive control result and be enough to upper condition, it is invalid that this time experiment is considered as.Under the prerequisite that detected result is set up, if sample detection result Ct value≤27.0, and there is specific amplification curve, be judged to be the positive; Ct value > 30.0 or nothing, and without specific amplification curve, be then judged to be feminine gender; 30.0 >=Ct value > 27.0 and to have the sample of specific amplification curve to be judged to be suspicious, again test suspicious specimen, result is that positive is judged to be the positive, and result is that negative patient is judged to be feminine gender; Again test suspicious specimen, result answers this animal sample of Resurvey again to test for suspicious person again.Same increment product judge: universal detection system FAM fluorescence channel and A type VIC fluorescence channel are positive and are judged to be that this sample is as the two positive of T/A type, and namely this sample is A type infection of foot-and-mouth disease sample; Universal detection system FAM fluorescence channel is positive, and A type detection system VIC fluorescence channel negative patient is judged to be that this sample is universal positive, and namely this sample is its alloytype infection of foot-and-mouth disease sample, and non-A type infection of foot-and-mouth disease sample.
Foot and mouth disease virus provided by the invention is universal, the double real-time fluorescence quantitative PCR of A type detects primer and probe, and to detect based on the double real-time fluorescence quantitative PCR that described primer and probe are set up and can realize a reaction and differentiate fast to detect to O, A, Asia I 3 serotype foot and mouth disease virus FMDV-T and A type foot and mouth disease virus FMDV-A simultaneously, detection can be completed in 1-2h, there is the advantages such as quick, special, responsive, high-throughput, requirement in enormous quantities, to differentiate to detect FMDV-T, FMDV-A fast can be met.
Accompanying drawing explanation
Fig. 1: the qualification of general FMDV recombinant plasmid; Wherein, M is DL2000Marker, and 1 is general FMDV recombinant plasmid pcr amplification product, and amplified production is 633bp.
The qualification of Fig. 2: A type FMDV recombinant plasmid; Wherein, M is 100bp Marker, and 1 is A type FMDV recombinant plasmid pcr amplification product, and amplified production is 326bp.
Fig. 3: the foundation of the double real-time fluorescence quantitative PCR diagnostic method of foot and mouth disease; Wherein, 1 is FAM probe FMDV-T amplification curve, and 2 is VIC probe FMDV-A amplification curve, and 3,4 is negative control.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 foot and mouth disease virus is general, the foundation of the double real-time fluorescence quantitative PCR detection system of A type
1. materials and methods
1.1 material
1.1.1 strain
O type, A type and Asia I type foot and mouth disease virus type strain, all purchased from Ministry of Agriculture's Lanzhou veterinary institute; Other contrast viruses or plasmid are preserved by animal epidemic prevention and control center, Henan Province.
1.1.2 instrument and reagent
Fluorescent PCR instrument, American AB I Products, model ABI7000; PCR amplification instrument, German Biometra Products; Labworks image acquisition and analysis software, U.S. Alpha Innotech Products; Thermostatic water bath vibrator (HZQ-Q), Harbin Dong Lian electronic technology development corporation, Ltd. product; Table-type high-speed refrigerated centrifuge, U.S. Heraeus Products; Ex Taq archaeal dna polymerase, dNTPs, DNA recovery test kit etc. are all purchased from precious biotechnology (Dalian) company limited, and pGEM-T Easy carrier, JM109 competent cell are purchased from Promega company.
1.2 method
1.2.1 design of primers and synthesis
The gene of FMDV 7 serotypes and FMDV-A gene order in a large amount of comparison GenBank, choose the O of FMDV, A, Asia I 3 serotype gene high conservative gene orders (SEQ ID NO:1) and FMDV-A gene high conservative and have Auele Specific Primer that type specificity gene order (SEQ ID NO:2) is stencil design FMDV-T/FMDV-A to TaqMan MGB probe, called after FMDVR (P0) respectively, FMDV-T (P1), FMDV-T (P2), FMDV-A (P3), FMDV-A (P4), FMDV-T-MGB-FAM-Probe (Probe-P5), FMDV-A-MGB-VIC-Probe (Probe-P6), synthesized by precious biotechnology (Dalian) company limited, for FMDV-T/FMDV-A double TaqMan MGB FQ-PCR increase primer and probe sequence as follows:
P0：5′-CGGGAAACGCATGAGCAGTA-3′
P1：5′-CGGCCTCTCATCCAACAGA-3′
P2：5′-ATTTTCCTTCAGGCGCTTGA-3′
P3：5′-AACCCCACCGCCTACCA-3′
P4：5′-GGTGTAAGGGAGCGCAAGTC-3′
Probe-P5：5'-FAM-TCATTTGTGAAACGCG-MGB-3'
Probe-P6：5'-VIC-AAGCAGCCGTTTACG-MGB-3'
1.2.2 the preparation of total serum IgE
The preparation of template ribonucleic acid: with FMDV-T/FMDV – A positive plasmid for positive control, water belongs with yin contrasts, and adopts Trizol method to extract total serum IgE with O type, A type and Asia I type foot and mouth disease virus type strain simultaneously.Concrete operations are as follows: get O type, A type and Asia I type foot and mouth disease virus type strain, negative control and each 200 μ L of measuring samples respectively in 1.5ml centrifuge tube, the Trizol adding 600 μ L again shakes 2-3min in vortice, add 200 μ L chloroforms, after centrifugal, getting supernatant proceeds in another 1.5ml centrifuge tube, add 200 μ L isopropanol precipitatings, 75% washing with alcohol precipitation, dry, finally use 20 μ L DEPC (tetra-sodium diethyl ester) water dissolution precipitations, get 10 μ L for reverse transcription, all the other-20 DEG C preservations.
1.2.3 reverse transcription
Often pipe FMDV reverse transcription reaction system is containing following composition: 5 × M-MLV damping fluid 4 μ L; 2.5mmol/L dNTPs (triphosphate deoxy-nucleotide) 4 μ L; M-MLV ThermoScript II 0.5 μ L; RNase inhibitor 0.5 μ L; Primer P01 μ L; Cumulative volume 10 μ L.In every pipe reverse transcription reaction system, add RNA 10 μ L obtained in step 1.2.2,37 DEG C of water-bath 1h or be placed in PCR instrument 37 DEG C reaction 1h, after reaction terminates, 70 DEG C, 15min deactivation ThermoScript II, be directly used in subsequent PCR amplification or-20 DEG C frozen for subsequent use.
1.2.4FMDV-T/FMDV-A the preparation of standard substance
The positive amplification product of FMDV-T/FMDV-A is cloned in pGEM-T Easy carrier respectively, screens positive recombinant plasmid and send precious biotechnology (Dalian) company limited to adopt T7 and SP6 primer to check order.Its OD of FMDV-T/FMDV-A restructuring positive plasmid application spectrophotometric determination checking order correct
260and OD
280value and OD
260/ OD
280value, repeats 5 times altogether; With reference to plasmid DNA copies number calculating method, the concentration calculating pGEM-T/FMDV-T and pGEM-T/FMDV-A plasmid DNA solution is respectively 9.12 × 10
10copy/μ L and 9.11 × 10
10copy/μ L, quantitatively and be diluted to 1.0 × 10
0~ 1.0 × 10
10copy/μ L ,-20 DEG C save backup.General FMDV recombinant plasmid (pGEM-T/FMDV-T) and A type FMDV recombinant plasmid (pGEM-T/FMDV-A) are identified.(as Fig. 1 and 2, the amplified production of general FMDV recombinant plasmid is the amplified production of 633bp, A type FMDV recombinant plasmid is 326bp.)
1.2.5FMDV-T/FMDV-A the optimization of double FQ-PCR reaction conditions
PGEM-T/FMDV-T and the pGEM-T/FMDV-A recombinant plasmid obtained in step 1.2.4 is diluted to final concentration 1.0 × 10 respectively
5copy/μ L is as detection template, P1/P2, P3/P4 primer pair dilutes for final concentration 0.05 respectively, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 μm of ol/L, Probe-P5, it is 0.05 that Probe-P6 probe is diluted to final concentration respectively, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 μm of ol/L, application fluorescent PCR instrument (American AB I company, model: ABI7000) adopt matrix method to screen FMDV-T/FMDV-A primer and the probe combinations of different concns, screen the best primer concentration of double FQ-PCR for FMDV-T/FMDV-A, concentration and probe concentration and optimum reaction condition.
The 25 μ L PCR reaction systems optimized, the each 0.4 μm of ol/L of FMDV-T/FMDV-A upstream and downstream primer P1/P2, P3/P4 final concentration, Probe-P5 final concentration is 0.6 μm of ol/L, Probe-P6 final concentration is 0.7 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, 10 × Ex Taq enzyme damping fluid 2.5 μ L, 5U/ μ L Ex Taq DNA enzymatic 0.25 μ L, 1.0 × 10
5the each 2 μ L of plasmid template of copy/μ L, supply deionized water to final volume 25 μ L.The PCR response procedures optimized is: 94 DEG C, 5min; 94 DEG C of 15s, 60 DEG C of 30s, 40 circulations (as Fig. 3,1 is FAM probe FMDV-T amplification curve, and 2 is VIC probe FMDV-A amplification curve, and 3,4 is negative control).
Result judges: threshold setting is as the criterion with the vertex of threshold line just above normal negative controls amplification curve, and different instrument can adjust according to instrument noise situation.The detected result of negative control should without specific amplification curve, and Ct value > 30.0 or nothing, and the Ct value of positive control should≤27.0, and occur specific amplification curve, judge that detected result is set up.Be discontented with as negative control and positive control result and be enough to upper condition, it is invalid that this time experiment is considered as.Under the prerequisite that detected result is set up, if sample detection result Ct value≤27.0, and there is specific amplification curve, be judged to be the positive; Ct value > 30.0 or nothing, and without specific amplification curve, be then judged to be feminine gender; 30.0 >=Ct value > 27.0 and to have the sample of specific amplification curve to be judged to be suspicious, again test suspicious specimen, result is that positive is judged to be the positive, and result is that negative patient is judged to be feminine gender; Again test suspicious specimen, result answers this animal sample of Resurvey again to test for suspicious person again.Universal detection system FAM fluorescence channel and A type VIC fluorescence channel are positive and are judged to be that this sample is the two positive of T/A type, and namely this sample is A type infection of foot-and-mouth disease sample; Universal detection system FAM fluorescence channel is positive, and A type detection system VIC fluorescence channel negative patient is judged to be that this sample is universal positive, and namely this sample is its alloytype infection of foot-and-mouth disease sample, and non-A type infection of foot-and-mouth disease sample.
1.2.6 the foundation of sensitivity test and typical curve
PGEM-T/FMDV-T and the pGEM-T/FMDV-A recombinant plasmid obtained in step 1.2.4 is made 10 times of serial dilutions respectively, and two kinds of plasmid 1:1 ratio mixing, often kind of plasmid final concentration is adjusted to 1.0 × 10
7~ 1.0 × 10
0copy/μ L, totally 8 extent of dilution take pure water as negative control, and the FQ-PCR reaction conditions optimized by step 1.2.5 carries out the double FQ-PCR sensitivity test of FMDV-T/FMDV-A.Respectively with the logarithm of pGEM-T/FMDV-T and pGEM-T/FMDV-A recombinant plasmid starting template number for X-axis, with FQ-PCR cycle index C
tvalue makes regression curve for Y-axis, sets up the typical curve of double FQ-PCR method.Double FQ-PCR detects FMDV-T and FMDV-A minimum detectability and is 1.0 × 10
1copy/μ L, experimental result is in table 1.
Table 1
1.2.7 specific test
By method described in step 1.2.2, RNA carry out reverse transcription by step 1.2.2 is extracted to Pestivirus suis (CSFV), PRRS virus (PRRSV) etc., the then extracting directly DNA such as PRV (Pseudorabies virus) (PRV), porcine circovirus 2 type (PCV), pig parvoviral (PPV), mycoplasma hyopneumoniae, is respectively 1.0 × 10 by concentration simultaneously
5pGEM-T/FMDV-T and the pGEM-T/FMDV-A recombinant plasmid mixture of copy/μ L is as positive control; high-pathogenicity blue ear disease vaccine is negative control, and the FQ-PCR reaction conditions optimized by step 1.2.5 carries out double FQ-PCR amplification to verify the specificity of the method.Ct value is respectively 13.5641 and 14.2809, and occurs specific amplification curve; But all there is not specific amplification curve in pure water, Pestivirus suis (CSFV), PRRS virus (PRRSV), PRV (Pseudorabies virus) (PRV), porcine circovirus 2 type (PCV), pig parvoviral (PPV).Experimental result is in table 2, and in table 2, each sample concentration is 1.0 × 10
7copy/μ L.
Table 2
1.2.8 stability and replica test
Respectively by pGEM-T/FMDV-T and pGEM-T/FMDV-A recombinant plasmid equal amount of mixture (often kind of plasmid final concentration 1.0 × 10 of 10 of 4 concentration times of serial dilutions
3~ 1.0 × 10
0) increase according to the FQ-PCR reaction conditions of step 1.2.5 optimization, each series setting 3 repetitions, to verify stability and the repeatability of the method.Replica test result shows, concentration is 1.0 × 10
3copy/μ L, 1.0 × 10
2copy/μ L, 1.0 × 10
1fMDV-T/FMDV-A recombinant plasmid equal amount of mixture 3 test-results of 3 concentration of copy/μ L are the positive, and concentration is 1.0 × 10
0fMDV-T/FMDV-A recombinant plasmid equal amount of mixture 3 test-results of copy/μ L are feminine gender, illustrate that the FMDV-T/FMDV-A double FQ-PCR method set up has good stability and repeatability.Detected result is in table 3.
Table 3
Embodiment 2 foot and mouth disease virus is general, the application of the double real-time fluorescence quantitative PCR detection system of A type
The FMDV-T/FMDV-A conventional RT-PCR detection method that the double FQ-PCR of FMDV-T/FMDV-A set up according to this research and this laboratory were once set up, preparation detection reagent, with FMDV-T/FMDV-A positive plasmid mixture for positive control, water belongs with yin contrasts, (sample number into spectrum is: 1 ~ 13) carried out applying detection to infect sample to 13 parts of clinical doubtful FMDV, first to 13 increment product, refiner grinding homogenate is carried out in yin and yang attribute contrast, centrifugal 5 minutes of 5000r/min, get supernatant 100 μ L, instrument for extracting nucleic acid extracts total serum IgE, utilize method provided by the present invention and foot and mouth disease virus O type, (conventional RT-PCR detects for A type and the triple RT-PCR detection reagent of Asia I and detection method thereof, Authorization Notice No. CN 102230029B) test in laboratory is carried out to each sample regulating YIN and YANG contrast, finally analysis is compared to the detected result of these two kinds of methods, and compare with sequencing result.Result shows, adopts FMDV-T/FMDV-A duplex PCR provided by the present invention to detect, and 2 parts is that FMDV-T is positive, and 1 part is that FMDV-T/FMDV-A is two positive; Adopt conventional RT-PCR to detect 1 part for the FMDV-T positive, 1 part is that FMDV-T/FMDV-A is two positive.Show that the FMDV-T/FMDV-A double FQ-PCR method that the present invention sets up is higher than the susceptibility of conventional RT-PCR method, this FQ-PCR method detected result and FMDV-T/FMDV-A FMDV gene sequencing result coincidence rate are 100%.Detected result is in table 4.
Table 4
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. the double real-time fluorescence quantitative PCR of general, the A type of foot and mouth disease virus detects primer and probe, and it is characterized in that, described primer comprises:
P0：5′-CGGGAAACGCATGAGCAGTA-3′
P1：5′-CGGCCTCTCATCCAACAGA-3′
P2：5′-ATTTTCCTTCAGGCGCTTGA-3′
P3：5′-AACCCCACCGCCTACCA-3′
P4：5′-GGTGTAAGGGAGCGCAAGTC-3′
Described probe comprises:
Probe-P5：5'-F1-TCATTTGTGAAACGCG-Q1-3'
Probe-P6：5'-F2-AAGCAGCCGTTTACG-Q2-3'
Wherein, F1, F2 are fluorophor, and Q1, Q2 are non-fluorescence quenching group.
2. primer according to claim 1 and probe, is characterized in that, F1 is FAM, F2 be VIC, Q1 and Q2 is MGB.
3. contain the test kit detecting foot and mouth disease virus and A type foot and mouth disease virus for real-time fluorescence quantitative PCR of primer described in claim 1 or 2 and probe.
4. primer described in claim 1 or 2 and probe, or test kit described in claim 3 detects the application in foot and mouth disease virus and A type foot and mouth disease virus at real-time fluorescence quantitative PCR.
5. application according to claim 4, is characterized in that, comprises the following steps:
1) extract the total serum IgE of viral sample to be measured, carry out reverse transcription and obtain cDNA; Wherein, reverse transcription system is: 5 × M-MLV damping fluid 4 μ L, 2.5mmol/L dNTP 4 μ L, primer P01 μ L, M-MLV ThermoScript II 0.5 μ L, RNase inhibitor 0.5 μ L, RNA 10 μ L; Reverse transcription condition is: 37 DEG C, 1h;
2) being cloned into by cDNA in pGEM-T Easy carrier, screening positive recombinant plasmid, take plasmid as template, carries out pcr amplification reaction;
3) PCR primer is analyzed.
6. application according to claim 5, it is characterized in that, the reaction system of carrying out pcr amplification reaction use is counted with 25 μ l: P1, P2, P3, P4 final concentration is respectively 0.4 μm of ol/L, Probe-P5 final concentration is 0.6 μm of ol/L, Probe-P6 final concentration is 0.7 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, 10 × Ex Taq enzyme damping fluid 2.5 μ L, 5U/ μ L Ex Taq DNA enzymatic 0.25 μ L, 1.0 × 10
5the plasmid template 2 μ L of copy/μ L, supplies deionized water to final volume 25 μ L.
7. application according to claim 5, is characterized in that, pcr amplification reaction condition is: 94 DEG C 5 minutes; 94 DEG C 15 seconds, 60 DEG C 30 seconds, totally 40 circulations.
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| CN105112558A (en) * | 2015-08-06 | 2015-12-02 | 河南省动物疫病预防控制中心 | Foot and mouth disease viruses O, A and Asia I type ternary real time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit |
| CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
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| CN103602757A (en) * | 2013-08-30 | 2014-02-26 | 天津出入境检验检疫局动植物与食品检测中心 | Development and application of multiple fluorescence RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and swine vesicular disease |
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| CN101928780A (en) * | 2009-06-25 | 2010-12-29 | 北京索奥生物医药科技有限公司 | A type foot-and-mouth disease virus two-color fluorescence PCR detection method and kit thereof |
| CN103602757A (en) * | 2013-08-30 | 2014-02-26 | 天津出入境检验检疫局动植物与食品检测中心 | Development and application of multiple fluorescence RT-PCR detection method for foot-and-mouth disease, vesicular stomatitis and swine vesicular disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105112558A (en) * | 2015-08-06 | 2015-12-02 | 河南省动物疫病预防控制中心 | Foot and mouth disease viruses O, A and Asia I type ternary real time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit |
| CN105112558B (en) * | 2015-08-06 | 2018-02-06 | 河南省动物疫病预防控制中心 | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I |
| CN107326100A (en) * | 2017-07-17 | 2017-11-07 | 河南省动物疫病预防控制中心 | Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus |
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