CN105112558A - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention provides a 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. The 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 is a common reverse transcription primer of three serotypes comprising a foot and mouth disease viruses O type, a foot and mouth disease viruses A type and a foot and mouth disease viruses Asia I type which are designed by foot and mouth disease viruses 2B genes; a foot and mouth disease viruses VP1 gene serves as an amplification target region, three pairs of foot and mouth disease viruses type specific primers and TaqMan MGB probs are designed, and the three serotypes comprising the foot and mouth disease viruses O type, the foot and mouth disease viruses A type and the foot and mouth disease viruses Asia I type are identified and detected by a real time fluorescent quantitative RT-PCR technology. The detection kit is suitable for detecting tissue samples such as blister skin, tonsilla and lymph nodes, and virus nucleic acids of blister fluid and oesophagus and pharyngeal secretion (O-P secretion) of suspected food and mouth disease viruses infected livestock, sensitivity can reach 1.0-101 copy/micro L, and the foot and mouth disease viruses O, A and Asia I type ternary real time fluorescent quantitative RT-PCR detection kit does not have cross reaction with other pathogens which are easily infected with foot and mouth disease viruses in a mixed manner or pathogens of which infection symptoms are similar to those of the foot and mouth disease viruses, such as VSV (vesicular stomatitis viruses), PRV (pseudorabies viruses), CSFV (classical swine fever viruses), PRRSV (porcine reproductive and respiratory syndrome viruses), SS2, GPV (goose plaque viruses) and BVDV (bovine viral diarrhea viruses).
Description
Technical field
The present invention relates to the molecular Biological Detection technology of foot and mouth disease virus, specifically, relate to foot and mouth disease virus O type, A type and the triple real-time fluorescence quantitative RT-PCR of Asia I type (FQ-PCR) detection kit.
Background technology
Foot and mouth disease is caused by foot and mouth disease virus (FMDV) that one artiodactylous is acute, hot, high degree in contact sexually transmitted disease, has significant damage to artiodactyl beast.Foot and mouth disease virus has O, A, C, SAT1, SAT2, SAT3 (i.e. South Africa 1,2,3 type) and Asia I (Asia I type) 7 serotypes, China is O, A and Asia I type mainly, can not produce cross immunity between the virus of each serotype.Therefore, foot and mouth disease virus fast and accurately somatotype is the important content of foot and mouth disease diagnosis, and the selection and the epidemiological study that can be vaccine provide valuable information.
Summary of the invention
The object of this invention is to provide the primer and probe that detect for foot and mouth disease virus O type, A type and the triple real-time fluorescence quantitative RT-PCR of Asia I type (FQ-PCR).
Another object of the present invention is to provide the triple FQ-PCR detection kit of foot and mouth disease virus O type, A type and Asia I type.
In order to realize the object of the invention, the primer for the triple FQ-PCR detection of foot and mouth disease virus O type, A type and Asia I type provided by the invention and probe, comprising:
O type FMDV detects primer and probe:
P1：5'-GSGCRCTCCTBCGNACK-3'
P2：5'-YRTGYTTSACWGCYASYTCYARR-3'
Probe-P7：5'-FAM-CYACYTACTAYTTCKCW-MGB-3'；
A type FMDV detects primer and probe:
P3：5'-CYGAGGCAGCACTGVAM-3'
P4：5'-CGGYGCYTTGTGGTAAGC-3'
Probe-P8：5'-VIC-CMCGAGCAACCCCA-MGB-3'；
Asia I type FMDV detects primer and probe:
P5：5'-AAGAGCACCCAAACCCTTGA-3'
P6：5'-GCCCCGACCAGTGTGTGT-3'
Probe-P9:5'-NED-CTCATGCAGATCCCCT-MGB-3'; And
Share reverse transcription primer P0:5'-CGGGAAACGCATGAGCAGTA-3';
Wherein, S is G, C; R is A, G; B is C, G, T; N is A, C, G, T; K is G, T; Y is C, T; W is A, T; V is A, C, G; M is A, C.
The present invention also provide containing described primer and probe for detecting O, A and Asia I triple real-time fluorescence quantitative RT-PCR test kit of type foot and mouth disease virus.
Preferably, described test kit also comprises dNTPs, reversed transcriptive enzyme, Taq DNA polymerase, Mg
2+, one or more in PCR reaction buffer etc.
More preferably, described test kit also comprises positive control and negative control.Wherein, described positive control is through the foot and mouth disease virus O type of deactivation, A type and Asia I type type strain cell culture; Described negative control is normal cell nutrient solution.Arranging O type, A type and Asia I type detection system is respectively FAM, VIC and NED fluorescence channel.
Reverse transcription system for described test kit is: 5 × M-MLV damping fluid 4 μ L, 2.5mmol/LdNTP4 μ L, and 20 μm of ol/L share reverse transcription primer P01 μ L, M-MLV ThermoScript II 0.5 μ L, RNase inhibitor 0.5 μ L, sample RNA10 μ L; Reverse transcription condition is: 37 DEG C, 1h.
Reaction system for fluorescence quantitative RT-RCR amplified reaction is counted with 25 μ l: primer P1, P2, P3, P4, P5, P6 final concentration is respectively 0.4 μm of ol/L, Probe-P7 final concentration is 0.4 μm of ol/L, Probe-P8 final concentration is 0.6 μm of ol/L, Probe-P9 final concentration is 0.6 μm of ol/L, 2.5mmol/LdNTPs2 μ L, 10 × ExTaq enzyme buffer liquid 2.5 μ L, 5U/ μ LExTaqDNA polysaccharase 0.25 μ L, cDNA template 4 μ L, deionized water complements to cumulative volume 25 μ L.
Amplification reaction condition is: 94 DEG C 5 minutes; 94 DEG C 15 seconds, 60 DEG C 30 seconds, totally 40 circulations.
The present invention further provides described test kit detecting foot and mouth disease virus and differentiating the application of O, A and Asia I in C-type virus C.Particularly, 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/LdNTP4 μ L, shares reverse transcription primer P01 μ L (20 μm of ol/L), M-MLV ThermoScript II 0.5 μ L, RNase inhibitor 0.5 μ L, RNA10 μ L; Reverse transcription condition is: 37 DEG C, 1h.
2) fluorescence quantitative RT-RCR amplified reaction is carried out; Wherein, reaction system is counted with 25 μ l: primer P1, P2, P3, P4, P5, P6 final concentration is respectively 0.4 μm of ol/L, Probe-P7 final concentration is 0.4 μm of ol/L, Probe-P8 final concentration is 0.6 μm of ol/L, Probe-P9 final concentration is 0.6 μm of ol/L, 2.5mmol/LdNTPs2 μ L, 10 × ExTaq enzyme buffer liquid 2.5 μ L, 5U/ μ LExTaqDNA polysaccharase 0.25 μ L, cDNA template 4 μ L, deionized water complements to cumulative volume 25 μ L;
Amplification reaction condition is: 94 DEG C 5 minutes; 94 DEG C 15 seconds, 60 DEG C 30 seconds, totally 40 circulations.
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, can adjust according to different instrument noise situation.The detected result of negative control should without specific amplification curve, and Ct value > 30.0 or without Ct; The Ct value of positive control answers≤25.0, and occurs specific amplification curve, judges 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:
1) if 1 specific amplification curve appears in the FQ-PCR reaction system passage of sample to be tested, and Ct value≤32.0, then can be judged to be to there is FMDV in sample.
2) if the FQ-PCR reaction system passage of sample to be tested is without specific amplification curve, and Ct value > 35.0 or without Ct, be then judged to be that FMDV is negative.
3) if 1 specific amplification curve appears in the FQ-PCR reaction system passage of sample to be tested, and 32.0 < Ct value≤35.0, then need duplicate test, reproducible results is that the positive is then judged to be the positive, otherwise is judged to be feminine gender.
4) if there is 1 specific amplification curve and Ct value≤32.0 under " FAM/NONE " marking mode, show in sample, to there is FMDV-O type; If there is 1 specific amplification curve and Ct value≤32.0 under " VIC/NONE " marking mode, show in sample, to there is FMDV-A type; If there is 1 specific amplification curve and Ct value≤32.0 under " NED/NONE " marking mode, show in sample, to there is FMDV-Asia I type.
The triple real-time fluorescence quantitative RT-PCR of foot and mouth disease virus O type provided by the invention, A type and Asia I type detects primer and probe, and can realize differentiating fast to detect to O, A, Asia I 3 serotype foot and mouth disease viruses in a reaction based on triple real-time fluorescence quantitative RT-PCR detection methods that described primer and probe are set up 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 O, A, Asia I 3 serotype foot and mouth disease viruses fast can be met.
The present invention is with the shared reverse transcription primer of foot and mouth disease virus 2B gene design foot and mouth disease virus O type, A type and Asia I type three serotypes; Again using VP 1 Gene of Foot-and-Mouth Disease virus as amplified target region, design 3 pairs of foot and mouth disease virus type-special primer and TaqManMGB probe, adopt real-time fluorescence quantitative RT-PCR, realize the detection to foot and mouth disease virus O type, A type and Asia I type three serotypes.Detection kit provided by the invention is applicable to that doubtful foot and mouth disease virus (FMD) infects the tissue sample such as blister skin, tonsilla, lymphoglandula of domestic animal and blister liquid, the viral nucleic acid of esophagus-pharyngeal juice (O-P liquid) detects, and sensitivity can reach 1.0 × 10
1copy/μ L, with other easily similar to its polyinfection or infection symptoms pathogenic agent, such as no cross reaction between pig vesicular stomatitis virus (VSV), PRV (Pseudorabies virus) (PRV), Pestivirus suis (CSFV), PRRS virus (PRRSV), streptococcus suis 2-type (SS2), capripox virus (GPV), bovine viral diarrhea virus (BVDV).
Accompanying drawing explanation
Fig. 1 is foot and mouth disease virus O type, A type and the triple real-time fluorescent quantitative RT-PCR method of Asia I type set up amplification curve; Wherein, 1 is FMDV-O as killed cells poison amplification curve; 2 is FMDV-A type as killed cells poison amplification curve; 3 is FMDV-Aisa I type as killed cells poison amplification curve; 4 ~ 10 is pig vesicular stomatitis virus (VSV), PRV (Pseudorabies virus) (PRV), Pestivirus suis (CSFV), PRRS virus (PRRSV), streptococcus suis 2-type (SS2), capripox virus (GPV), bovine viral diarrhea virus (BVDV) amplification curve, and 11 is negative control amplification curve.
Fig. 2 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects O type foot and mouth disease virus susceptibility amplification curve; 1 ~ 6 corresponding respectively plasmid concentration is 1.0 × 10
0, 1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5copy/μ L; 7 is negative control.
Fig. 3 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects O type foot and mouth disease virus typical curve.
Fig. 4 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects A type foot and mouth disease virus susceptibility amplification curve; 1 ~ 6 corresponding respectively plasmid concentration is 1.0 × 10
0, 1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5copy/μ L; 7 is negative control.
Fig. 5 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects A type foot and mouth disease virus typical curve.
Fig. 6 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects Asia I type foot and mouth disease virus susceptibility amplification curve; 1 ~ 6 corresponding respectively plasmid concentration is 1.0 × 10
0, 1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5copy/μ L; 7 is negative control.
Fig. 7 is that the triple FQ-PCR method of foot and mouth disease virus O type, A type and Asia I type detects Asia I type foot and mouth disease virus typical curve.
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 (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 is for the triple primer of real-time fluorescence quantitative RT-PCR detection of foot and mouth disease virus O type, A type and Asia I type and the foundation of probe and detection method
1.1 material
1.1.1 strain
Through the foot and mouth disease virus O type of deactivation, A type and Asia I type type strain cell culture, from the foot and mouth disease detection kit of certain production of units domestic; Other contrast viruses, bacterium or positive recombinant plasmid are provided 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. AlphaInnotech 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; ExTaqDNA polysaccharase, dNTPs, enzyme inhibitors etc. are all purchased from precious biotechnology (Dalian) company limited, and M-MLV ThermoScript II, pGEM-TEasy carrier etc. are purchased from Promega company.
1.2 method
1.2.1 design of primers and synthesis
The gene of FMDV7 serotype in a large amount of comparison GenBank, choose the O of FMDV, A, Asia I 3 serotype gene high conservative 2B gene orders design 3 serotypes shared reverse transcription primer FMDV-R (P0) and based on O, A, the VP1 gene design Auele Specific Primer that Asia I 3 serotypes have a type specificity to TaqManMGB probe, called after FMD-O-P1 (P1) respectively, FMD-O-P2 (P2), FMD-A-P3 (P3), FMD-A-P4 (P4), FMD-Asia I-P5 (P5), FMD-Asia I-P6 (P6), FMD-O-MGB-FAM (Probe-P7), FMD-A-MGB-VIC (Probe-P8), FMD-Asia I-MGB-NED (Probe-P9).
Designed primer and probe sequence as follows:
O type FMDV detects primer and probe:
P1：5'-GSGCRCTCCTBCGNACK-3'
P2：5'-YRTGYTTSACWGCYASYTCYARR-3'
Probe-P7：5'-FAM-CYACYTACTAYTTCKCW-MGB-3'；
A type FMDV detects primer and probe:
P3：5'-CYGAGGCAGCACTGVAM-3'
P4：5'-CGGYGCYTTGTGGTAAGC-3'
Probe-P8：5'-VIC-CMCGAGCAACCCCA-MGB-3'；
Asia I type FMDV detects primer and probe:
P5：5'-AAGAGCACCCAAACCCTTGA-3'
P6：5'-GCCCCGACCAGTGTGTGT-3'
Probe-P9:5'-NED-CTCATGCAGATCCCCT-MGB-3'; And
Share reverse transcription primer P0:5'-CGGGAAACGCATGAGCAGTA-3';
Wherein, S is G, C; R is A, G; B is C, G, T; N is A, C, G, T; K is G, T; Y is C, T; W is A, T; V is A, C, G; M is A, C.
1.2.2 the preparation of total serum IgE
The preparation of template ribonucleic acid: with through the foot and mouth disease virus O type of deactivation, A type and Asia1 type type strain cell culture for positive control, with normal cell nutrient solution for negative control, adopt Trizol method to extract total serum IgE.
Concrete operations are as follows: get each 200 μ L of positive control, negative control and testing sample 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, get supernatant and proceed in another 1.5ml centrifuge tube, add 200 μ L isopropanol precipitatings, 75% washing with alcohol precipitation, dry, finally use 20 μ LDEPC (tetra-sodium diethyl ester) water dissolution to precipitate, get 10 μ L for reverse transcription, surplus is in-20 DEG C of 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/LdNTPs4 μ L; M-MLV ThermoScript II 0.5 μ L; RNase inhibitor 0.5 μ L; Share reverse transcription primer P01 μ L (20 μm of ol/L); Cumulative volume 10 μ L.In every pipe reverse transcription reaction system, add RNA10 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.
1.2.4pGEM-FMDV-O/pGEM-FMDV the preparation of – A/pGEM-FMDV – Asia I standard substance
Positive amplification product containing foot and mouth disease virus O type, A type and Asia I type VP1 gene is cloned in pGEM-TEasy carrier respectively, screens positive recombinant plasmid and send precious biotechnology (Dalian) company limited, adopt T7 and SP6 primer to check order.Check order correct O type, A type and its OD of the positive recombinant plasmid of Asia I (respectively called after pGEM-FMDV-O, pGEM-FMDV – A, pGEM-FMDV – Asia I) application spectrophotometric determination
260and OD
280value and OD
260/ OD
280value, repeats 5 times altogether, and with reference to plasmid DNA copies number calculating method, the concentration of calculating pGEM-FMDV-O, pGEM-FMDV – A, pGEM-FMDV – Asia I plasmid DNA solution is respectively 8.32 × 10
10copy/μ L, 8.81 × 10
10copy/μ L and 8.54 × 10
10copy/μ L, and be quantitatively diluted to 1.0 × 10 respectively
0~ 1.0 × 10
10copy/μ L ,-20 DEG C save backup.
1.2.5FMDVO the optimization of the triple FQ-PCR reaction conditions of type, A type and Asia I type
(1) pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I 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, P5/P6 primer pair is diluted to 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-P7, Probe-P8, it is 0.05 that Probe-P9 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 pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I primer and the probe combinations of different concns, screening is for FMDVO type, A type and the best primer concentration of the triple FQ-PCR of Asia I type, concentration and probe concentration and optimum reaction condition.
(2) in the 25 μ LPCR reaction systems optimized, FMDVO type, A type and each 0.4 μm of ol/L of Asia I type upstream and downstream primer P1/P2, P3/P4, P5/P6 final concentration, Probe-P7 final concentration is 0.4 μm of ol/L, Probe-P8 final concentration is 0.6 μm of ol/L, Probe-P9 final concentration is 0.6 μm of ol/L, 2.5mmol/L, dNTPs2 μ L, 10 × ExTaq enzyme buffer liquid 2.5 μ L, 5U/ μ LExTaqDNA polysaccharase 0.25 μ L, 1.0 × 10
5the each 2 μ L of plasmid template of copy/μ L, deionized water complements to cumulative 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 (Fig. 1).
(3) reaction parameter is arranged
1) probe in detecting pattern is set to: fluorescence mode FMDV-O type is set to FAM/NONE double-tagging pattern, and FMDV-A type is set to VIC/NONE double-tagging pattern, and FMDV-Asia I type is set to NED/NONE double-tagging pattern.
2) reaction conditions is set to: the first stage, denaturation 94 DEG C of 5min, 1 circulation; Subordinate phase, 94 DEG C of 15s, 60 DEG C of 1min, 40 circulations, carry out fluorescent signal detection at the end of the extension of each circulation.
3) quality control:
Negative control: the detected result under " FAM/NONE ", " VIC/NONE " and " NED/NONE " marking mode should be amplification curve without increased logarithmic phase, and Ct value > 35.0 or without Ct.
Positive control: occur 3 amplification curves having obvious increased logarithmic phase under " FAM/NONE ", " VIC/NONE " and " NED/NONE " marking mode, and Ct value answers≤25.0.
Above requirement need meet in once testing simultaneously, otherwise this experiment is invalid, need re-start.
4) result judges:
If 1. there is 1 specific amplification curve in the FQ-PCR reaction system passage of sample to be tested, and Ct value≤32.0, then can be judged to be to there is FMDV in sample.
If 2. the FQ-PCR reaction system passage of sample to be tested is without specific amplification curve, and Ct value > 35.0 or without Ct, be then judged to be that FMDV is negative.
If 3. there is 1 specific amplification curve in the FQ-PCR reaction system passage of sample to be tested, and 32.0 < Ct value≤35.0, then need duplicate test, reproducible results is that the positive is then judged to be the positive, otherwise is judged to be feminine gender.
If 4. there is 1 specific amplification curve and Ct value≤32.0 under " FAM/NONE " marking mode, show in sample, to there is FMDV-O type; If there is 1 specific amplification curve and Ct value≤32.0 under " VIC/NONE " marking mode, show in sample, to there is FMDV-A type; If there is 1 specific amplification curve and Ct value≤32.0 under " NED/NONE " marking mode, show in sample, to there is FMDV-Asia I type.
1.2.6 the foundation of sensitivity test and typical curve
The positive recombinant plasmid of pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I obtained in step 1.2.4 is carried out 10 times of serial dilutions respectively, and 3 kinds of plasmids are in the mixing of 1:1:1 ratio, and 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 triple FQ-PCR sensitivity test of FMDVO type, A type and Asia I type.Respectively with the positive recombinant plasmid initial template concentration of pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I for X-axis, with FQ-PCR cycle index C
tvalue makes regression curve for Y-axis, sets up the typical curve of triple FQ-PCR method.The minimum detectability that triple FQ-PCR detects the positive recombinant plasmid of pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I is 1.0 × 10
1copy/μ L.
FMDV-O type typical curve is set up, coefficient R by sensitivity Detection result
2be 0.9984, slope is-3.2509, and intercept is 36.481, thus draws the linear relationship expression formula between copy number (X) and Ct value: y=-3.2509logX+36.481
FMDV-A type typical curve is set up, coefficient R by sensitivity Detection result
2be 0.9949, slope is-3.2086, and intercept is 37.147, thus draws the linear relationship expression formula between copy number (X) and Ct value: y=-3.2086logX+37.147
FMDV-Asia I type typical curve is set up, coefficient R by sensitivity Detection result
2be 0.9947, slope is-3.3106, and intercept is 37.512, thus draws the linear relationship expression formula between copy number (X) and Ct value: y=-3.3106logX+37.512
The initial copy number (Fig. 2 ~ Fig. 7) that expression formula just can calculate FMDVO type, A type and Asia I type is substituted into according to the Ct value that instrument reads.
1.2.7 specific test
By method described in step 1.2.2 to Pestivirus suis (CSFV), PRRS virus (PRRSV), pig vesicular stomatitis virus (VSV) etc. extract RNA and carry out reverse transcription by step 1.2.2, PRV (Pseudorabies virus) (PRV), streptococcus suis 2-type (SS2), the then extracting directly DNA such as capripox virus (GPV), simultaneously by the foot and mouth disease virus O type through deactivation, A type and Asia I type type strain cell culture are positive control, with normal cell nutrient solution for negative control, reverse transcription is carried out by step 1.2.2, the FQ-PCR reaction conditions that step 1.2.5 optimizes carries out triple FQ-PCR amplification to verify the specificity of the method.Result shows that the foot and mouth disease virus O type of deactivation, A type and the Asia I type type strain cell culture positive control FQ-PCR Ct value that increases is respectively 17.56,17.84 and 18.76, and occurs specific amplification curve; But all there is not specific amplification curve in pig vesicular stomatitis virus (VSV), PRV (Pseudorabies virus) (PRV), Pestivirus suis (CSFV), PRRS virus (PRRSV), streptococcus suis 2-type (SS2), capripox virus (GPV), bovine viral diarrhea virus (BVDV), normal cell nutrient solution etc.Experimental result is shown in Fig. 1.
1.2.8 stability and replica test
Respectively by the positive recombinant plasmid equal amount of mixture of pGEM-FMDV-O/pGEM-FMDV – A/pGEM-FMDV – Asia I (often kind of plasmid final concentration 1.0 × 10 of 10 of 4 concentration times of serial dilutions
5~ 1.0 × 10
2) increase according to the FQ-PCR reaction conditions of step 1.2.5 optimization, each series setting 4 repetitions, are divided into stability and repeatability that 3 batches are carried out verifying the method.Result shows, in batch, the revision test variation coefficient (CV) is 1.16%, between batch, revision test CV is 1.51%, and both CV values, all below 3%, show that FMDVO type, A type and the Asia I type triple FQ-PCR method set up has good stability and repeatability.Test-results is in table 1.
Table 1 stability and replica test result
Embodiment 2 foot and mouth disease virus O type, A type and the triple real-time fluorescence quantitative RT-PCR of Asia I type (FQ-PCR) detection kit
Triple FQ-PCR detection kit of the present invention comprises that O type FMDV detects primer pair and probe, A type FMDV detects primer pair and probe, Asia I type FMDV detect primer pair and probe and 1 shared reverse transcription primer.
O type FMDV detects primer and probe:
P1：5'-GSGCRCTCCTBCGNACK-3'
P2：5'-YRTGYTTSACWGCYASYTCYARR-3'
Probe-P7：5'-FAM-CYACYTACTAYTTCKCW-MGB-3'
A type FMDV detects primer and probe:
P3：5'-CYGAGGCAGCACTGVAM-3'
P4：5'-CGGYGCYTTGTGGTAAGC-3'
Probe-P8：5'-VIC-CMCGAGCAACCCCA-MGB-3'
Asia I type FMDV detects primer and probe:
P5：5'-AAGAGCACCCAAACCCTTGA-3'
P6：5'-GCCCCGACCAGTGTGTGT-3'
Probe-P9：5'-NED-CTCATGCAGATCCCCT-MGB-3'
Share reverse transcription primer P0:5'-CGGGAAACGCATGAGCAGTA-3'
Wherein, S is G, C; R is A, G; B is C, G, T; N is A, C, G, T; K is G, T; Y is C, T; W is A, T; V is A, C, G; M is A, C.
Described detection kit also comprises dNTPs, reversed transcriptive enzyme, Taq DNA polymerase, Mg
2+, one or more in PCR reaction buffer etc.
The embody rule of described detection kit is as follows:
(1) sample requires:
1, apply Correct Technique and collect sample.
2, the throw out in sample and suspended substance may affect test-results, should centrifugal removing.
3, may not exceed 6 hours in room temperature placement after sample process, collection; If not detecting in 6 hours to be placed in the refrigerator of 2 ~ 8 DEG C by sample; Within more than 24 hours, preserve or transport if need, then should be frozen in less than-20 DEG C, avoid multigelation.Return to room temperature before using, shake mixing gently.
(2) method of inspection:
1, sample process
Blister liquid and the centrifugal rear 100 μ L supernatant liquors of directly getting of esophagus-pharyngeal juice (O-P liquid) sample are that sample to be tested uses; The tissue samples such as blister skin, tonsilla, lymphoglandula are got about 0.5g and are fully ground, and add 0.3mLPBS (pH7.4) the fully rear centrifugal 8-10min of 10000r/min of mixing, getting supernatant liquor is sample to be tested; The liquid samples such as cell culture can dilute or directly use as sample to be tested.
2, nucleic acid extraction
Business-like RNA rapid extraction test kit (centrifugal pillar) product can be adopted, operate according to test kit specification sheets; Or, business-like Full automatic instrument for extracting nucleic acid can be adopted, operate according to each producer instrument and matched reagent box specification sheets.
3, the reverse transcription of RNA
Getting RNA10 μ L adds in 10 μ L reverse transcription reaction systems, after 37 DEG C of water-bath 1h, the cDNA of acquisition can be directly used in Fluorescence PCR or-20 DEG C frozen for subsequent use.
Prepared by reverse transcription system: according to detection sample size according to single reaction 10 μ l consumption, calculate total amount needed for each component, after mixing, packing 10 μ l is in single PCR reaction tubes.Consider the loss caused in point process of assembling, the consumption detecting system needed for sample size more than reality can be prepared.
4, pcr amplification
Fluorescence PCR system mixed in advance to be divided and is filled in Fluorescence PCR pipe by 4.1, often pipe packing 21 μ L.
4.2 add positive control, negative control, sample to be tested cDNA each 4 μ L, 8000r/min centrifugal several seconds respectively in above-mentioned PCR reaction tubes, put into PCR amplification instrument.
Prepared by Fluorescence PCR system: according to detection sample size according to single reaction 21 μ l consumption, calculate total amount needed for each component, after mixing, packing 21 μ l is in single PCR reaction tubes.Consider the loss caused in point process of assembling, the consumption detecting system needed for sample size more than reality can be prepared.
5, reaction parameter is arranged
5.1 probe in detecting patterns are set to: fluorescence mode FMDV-O type is set to FAM/NONE double-tagging pattern, and FMDV-A type is set to VIC/NONE double-tagging pattern, and FMDV-Asia I type is set to NED/NONE double-tagging pattern.
5.2 reaction conditionss are set to: the first stage, denaturation 94 DEG C of 5min, 1 circulation; Subordinate phase, 94 DEG C of 15s, 60 DEG C of 1min, 40 circulations, carry out fluorescent signal detection at the end of the extension of each circulation.
(3) quality control:
(i) negative control: the detected result under " FAM/NONE ", " VIC/NONE " and " NED/NONE " marking mode should be amplification curve without increased logarithmic phase, and Ct value > 35.0 or nothing.
(ii) positive control: occur 3 amplification curves having obvious increased logarithmic phase under " FAM/NONE ", " VIC/NONE " and " NED/NONE " marking mode, and Ct value answers≤25.0.
Above requirement need meet in once testing simultaneously, otherwise this experiment is invalid, need re-start.
(4) result judges:
1) if 1 specific amplification curve appears in the FQ-PCR reaction system passage of sample to be tested, and Ct value≤32.0, then can be judged to be to there is FMDV in sample.
2) if the FQ-PCR reaction system passage of sample to be tested is without specific amplification curve, and Ct value > 35.0 or nothing, be then judged to be that FMDV is negative.
3) if 1 specific amplification curve appears in the FQ-PCR reaction system passage of sample to be tested, and 32.0 < Ct value≤35.0, then need duplicate test, reproducible results is that the positive is then judged to be the positive, otherwise is judged to be feminine gender.
4) if there is 1 specific amplification curve and Ct value≤32.0 under " FAM/NONE " marking mode, show in sample, to there is FMDV-O type; If there is 1 specific amplification curve and Ct value≤32.0 under " VIC/NONE " marking mode, show in sample, to there is FMDV-A type; If there is 1 specific amplification curve and Ct value≤32.0 under " NED/NONE " marking mode, show in sample, to there is FMDV-Asia I type.
The application of embodiment 3 foot and mouth disease virus O type, A type and the triple real-time fluorescence quantitative RT-PCR of Asia I type (FQ-PCR) detection technique
The foot and mouth disease virus O type set up according to the present invention, A type and the triple FQ-PCR of Asia I type and the foot and mouth disease virus O type set up before, A type and the triple RT-PCR detection method of Asia I type, preparation detection reagent, with through the foot and mouth disease virus O type of deactivation, A type and Asia I type type strain cell culture for positive control; With normal cell nutrient solution for negative control, by the domestic laboratory with foot and mouth disease virus separation and Culture research qualification to the clinical doubtful FMDV of its 15 parts of preserving infect sample (sample number into spectrum for: 1 ~ 15) adopt method disclosed in method provided by the invention and ZL201110163932.4 (conventional RT-PCR detection) to carry out test in laboratory respectively, and analysis is compared to the detected result of these two kinds of methods, and compare with sequencing result, the results are shown in Table 2.Result shows, adopts foot and mouth disease virus O type provided by the invention, A type and the triple FQ-PCR method of Asia I type to detect, and 4 parts is that FMDV-O is positive, and 2 parts is that FMDV-A is positive, and 1 part is that FMDV-Asia I is positive; Adopt conventional RT-PCR detect 3 parts for FMDV-O positive, 2 parts be FMDV-A positive and 1 part be FMDV-Asia I positive.Show that foot and mouth disease virus O type that the present invention sets up, A type are higher than the susceptibility of conventional RT-PCR method with Asia I type triple FQ-PCR method, this FQ-PCR method detected result and FMDV-O type/FMDV-A type/FMDV-Asia I type gene sequencing result coincidence rate are 100%.
Table 2 the inventive method compares with conventional RT-PCR and sequencing result
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 (5)
1., for primer and the probe of the triple real-time fluorescence quantitative RT-PCR detection of foot and mouth disease virus O type, A type and Asia I type, it is characterized in that, described primer and probe comprise:
O type FMDV detects primer and probe:
P1：5'-GSGCRCTCCTBCGNACK-3'
P2：5'-YRTGYTTSACWGCYASYTCYARR-3'
Probe-P7：5'-FAM-CYACYTACTAYTTCKCW-MGB-3'；
A type FMDV detects primer and probe:
P3：5'-CYGAGGCAGCACTGVAM-3'
P4：5'-CGGYGCYTTGTGGTAAGC-3'
Probe-P8：5'-VIC-CMCGAGCAACCCCA-MGB-3'；
Asia I type FMDV detects primer and probe:
P5：5'-AAGAGCACCCAAACCCTTGA-3'
P6：5'-GCCCCGACCAGTGTGTGT-3'
Probe-P9:5'-NED-CTCATGCAGATCCCCT-MGB-3'; And
Share reverse transcription primer P0:5'-CGGGAAACGCATGAGCAGTA-3';
Wherein, S is G, C; R is A, G; B is C, G, T; N is A, C, G, T; K is G, T; Y is C, T; W is A, T; V is A, C, G; M is A, C.
2. containing primer described in claim 1 and probe for detecting O, A and Asia I triple real-time fluorescence quantitative RT-PCR test kit of type foot and mouth disease virus.
3. test kit according to claim 2, is characterized in that, described test kit also comprises dNTPs, reversed transcriptive enzyme, Taq DNA polymerase, Mg
2+, one or more in PCR reaction buffer.
4. the test kit according to Claims 2 or 3, it is characterized in that, reverse transcription system for described test kit is: 5 × M-MLV damping fluid 4 μ L, 2.5mmol/LdNTP4 μ L, 20 μm of ol/L share reverse transcription primer P01 μ L, M-MLV ThermoScript II 0.5 μ L, RNase inhibitor 0.5 μ L, sample RNA10 μ L; Reverse transcription condition is: 37 DEG C, 1h.
5. the test kit according to Claims 2 or 3, it is characterized in that, reaction system for fluorescence quantitative RT-RCR amplified reaction is counted with 25 μ l: primer P1, P2, P3, P4, P5, P6 final concentration is respectively 0.4 μm of ol/L, Probe-P7 final concentration is 0.4 μm of ol/L, Probe-P8 final concentration is 0.6 μm of ol/L, Probe-P9 final concentration is 0.6 μm of ol/L, 2.5mmol/LdNTPs2 μ L, 10 × ExTaq enzyme buffer liquid 2.5 μ L, 5U/ μ LExTaqDNA polysaccharase 0.25 μ L, cDNA template 4 μ L, deionized water complements to cumulative volume 25 μ L;
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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| CN110964856A (en) * | 2019-12-23 | 2020-04-07 | 广西壮族自治区动物疫病预防控制中心 | Multiplex fluorescence quantitative RT-PCR kit for simultaneously detecting SVA and FMDV and detection method |
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