CN102230029B - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention belongs to the technical field of animal epidemic disease inspection and quarantine, in particular discloses a 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. The ternary RT-PCR detection reagent of the type O, type A and type Asial foot and mouth disease viruses, disclosed by the invention, comprises a common inverse transcription primer and three pairs of specific primers, wherein lengths of amplified target fragments are respectively 234 bp, 326 bp and 467 bp. The ternary RT-PCR detection method comprises the following method establishment related steps, like primer sequence, preparation of total RNA (Ribose Nucleic Acid), reverse transcription, PCR (Polymerase Chain Reaction) amplification and identification, result determination and the like. According to the invention, three serotypes of the foot and mouth disease virus can be simultaneously detected through a PCR reaction; the rapid and specific serotype diagnosis can be carried on the foot and mouth disease pathogen; and the rapid and specific method with high sensitivity has important significance in clinically rapid differential diagnosis of type O, type A and type Asial foot and mouth disease viruses.
Description
Technical field
The invention belongs to animal epidemic and detect the Quarantine Techniques field, relate to foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection reagent of type and detection method thereof.
Background technology
Foot and mouth disease (Foot and Mouth Disease, FMD) be by foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) cause take artiodactyl as main acute, hot, hyperinfection epidemic disease, OIE (OIE) classifies it as must report zoonosis, and China is defined as a class animal epidemic.
Foot and mouth disease virus can make many animals ill, mainly encroaches on the artiodactyl beast, with the susceptible of ox, is secondly pig, is the animals such as sheep, goat and camel again.The foot and mouth disease velocity of propagation is fast, and scope is wide, is to spread formula great-jump-forward is also arranged, and generally is endemicity, also can form periodically to be very popular.Under state of nature, several infectious viral particles can cause the livestock morbidity, and the epidemic-stricken area sickness rate can reach 50～100%, and the cub mortality ratio is higher, and other adult animals are lower.After livestock infects foot and mouth disease, can cause livestock to stop appetite and descend, Abwehrkraft des Koepers and immunizing power reduce, easily other virus diseases of secondary and bacteriosis, thus cause the production performance degradation of infection animal.Along with the development of China's breeding scale industry, the foot and mouth disease velocity of propagation is more and more faster, and scope is also in continuous expansion, and the continuous change of epidemic isolates serotype in addition makes foot and mouth disease control difficulty and continues to increase.At present, China's a large amount of human and material resources of every average annual cost and financial resources are controlled and are eliminated, but should disease still happen occasionally every year.
Foot and mouth disease is divided O type, A type, C type, South Africa 1 type, South Africa 2 types, South Africa 3 types, 7 serotypes of Asia 1 type (Asia I type), each principal mode divides again some hypotypes, between each principal mode without cross-immunity, and can propagate between different animal species, also only have part that Immunogenicity is arranged between the different subtype in same principal mode.China has the case of pig, cattle infected O type and Asia 1 type foot and mouth disease at present, since in January, 2009, and the existing many cases report that infects A type foot and mouth disease of China milk cow, so A type foot and mouth disease is also very large to the potential hazard of pig.The threat of Asia 1, O, three kinds of serotype foot and mouth disease of A will be faced in mass-producing livestock field, and the prevention and control difficulty further strengthens.
At present existing Asia 1 type foot and mouth disease LPB-ELISA method, O type foot and mouth disease forward indirect hemagglutination test or LPB-ELISA method, Nonstructural Protein (NSPs) indirect ELISA method etc., these serological methods can only be estimated the single serotype antibody horizontal of foot and mouth disease.The RT-PCR diagnostic method has the advantage such as quick, special, responsive has been used widely in the epidemic disease etiological diagnosis, becomes one of animal epidemic etiology fast diagnosis method.Although domestic existing investigator has set up the single serotype foot and mouth disease RT-PCR diagnostic method for O type, A type and Asia 1 type, can not detect three kinds of serotypes simultaneously.Share reverse transcription primer and type specificity primer for foot and mouth disease virus O type, A type and three kinds of serotype designs of Asia 1 type, set up foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type, can differentiate three kinds of serotype foot and mouth disease cause of diseases simultaneously through a PCR reaction and make a definite diagnosis, method is quick, special, susceptibility is high, for foot and mouth disease virus O type, A type and Asia 1 type rapid differential diagnosis are significant clinically, yet there are no relevant report.
Summary of the invention
The present invention has set up foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection reagent of type and detection method thereof, purpose be to three kinds of serotypes of foot and mouth disease cause of disease carry out fast, classification diagnosis specifically, in order to take measures targetedly to carry out prevention and control.
For realizing purpose of the present invention, common reverse transcription primer is adopted in this research, three pairs of specific PCR primers are set up foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR of type, optimizing reaction system component, reaction conditions, carry out specificity, sensitivity, repeatability checking, utilize the foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type that are successfully established to carry out Preliminary Applications and detect.Main contents are as follows:
1. primer sequence
Foot and mouth disease virus O type, A type and Asia 1 type complete genome sequence in a large amount of comparison GenBank, the 2B gene of selecting foot and mouth disease virus O type, A type and 1 three serotype high conservatives of Asia is that stencil design shares reverse transcription primer P0, according to these 3 serotype type specificity VP1 gene orders, design trizygoid Auele Specific Primer (P1/P2, P3/P4, P5/P6), synthetic by Dalian (treasured) biotechnology company limited, being diluted to final concentration is 20 μ mol/ L.Be used for foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR primer sequences of type as follows:
P0：tgtcctcctg catctggttg 20
P
1：gtccagagac gccaacacac g 21
P
2：ggtgttgtcc aacgctgtct cg 22
P
3：agccccacgc acgtcattga c 21
P
4：acccgcgccg cgagagacc 19
P
5：agcccaagag cacccaaacc c 21
P
6：acggcggtct tgtgtggtgt c 21
2. the preparation of template ribonucleic acid
Single and the positive contrast of equal amount of mixture through the foot and mouth disease virus O of deactivation type, A type and Asia 1 type type strain cell culture, the normal negative contrast of BHK-21 cell adopt the Trizol method to extract total RNA with detected sample simultaneously.concrete operations are as follows: get respectively inactivation of viruses cell culture monoculture and equal amount of mixture, each 200 μ L of negative control and sample to be checked (getting supernatant after appropriate PBS grinds as first adding when organizing) are in the 1.5mL centrifuge tube, add again the Trizol of 600 μ L to shake 2-3min in vortice, add 200 μ L chloroforms, after centrifugal, getting supernatant changes in another 1.5mL centrifuge tube, add 200 μ L isopropanol precipitatings, the washing with alcohol precipitation of mass percent 75%, dry, use at last 20 μ L DEPC(diethylpyrocarbonates) the water dissolution precipitation, get 10 μ L and be used for reverse transcription, all the other-20 ℃ of preservations.
Reverse transcription
Every pipe reverse transcription reaction system contains 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; RNA enzyme inhibitors 0.5 μ L; P0 primer 1 μ L, cumulative volume 10 μ L.Add respectively the prepared RNA 10 μ L of step 2 in every pipe reverse transcription reaction system, 37 ℃ of water-bath 1h or be placed in 37 ℃, PCR instrument reaction 1h, after reaction finishes, 70 ℃, 15 min deactivation ThermoScript II, be directly used in following pcr amplification or-20 ℃ frozen standby.
Pcr amplification and evaluation
Carry out the triple PCR amplification take reverse transcription product as template.By the optimization to PCR reaction system and reaction conditions, determine that reaction system is as follows: adopt 25 μ L reaction systems, wherein: 10 *
ExTaqDamping fluid 2.5 μ L, 2.5mM dNTPs 2 μ L,
The ExTaq polymerizationEnzyme 2.5 U, each 0.5 μ L of 3 pairs of type specificity primers (P1/P2, P3/P4, P5/P6), the single cell culture of the prepared O type of step 3, A type and Asia 1 type and equal amount of mixture, normal each 2 μ L of BHK-21 cell reverse transcription product, mend to 25 μ L with deionized water, put on the PCR instrument and react.Amplification condition is 94 ℃, after 5min, and 94 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations, last 72 ℃ are extended 10min.The PCR product is through 1.5 % agarose electrophoresis observationss.
The product fragment of foot and mouth disease virus O type, A type and the positive amplification of Asia 1 type type strain is cloned into respectively pGEM-T easy carrier, screens positive recombinant plasmid and send Dalian precious biotechnology company limited to adopt T7 and SP6 primer to check order.
Specific test
By the described method of step 2 to extraction RNA such as swine vesicular disease virus, pig breeding and breathing syndrome virus, pig vesicular stomatitis virus, Pestivirus suis and carry out reverse transcription by step 3, PRV (Pseudorabies virus), streptococcus suis 2-type and toxoplasma gondii etc. directly extract DNA, establish simultaneously through the foot and mouth disease virus O of deactivation type, A type and the Asia 1 positive contrast of type type strain cell culture equal amount of mixture, the normal negative contrast of BHK-21 cell cDNA is carried out pcr amplification with the specificity of checking the method according to the described method of step 4.Carried out following proof test:
(a) sensitivity test
Foot and mouth disease virus O type, A type and the Asia 1 type pGEM-T easy recombinant plasmid of step 4 gained are made respectively 10 times of serial dilutions, and three kinds of plasmid 1:1:1 ratios are mixed, and every kind of plasmid final concentration is adjusted into 1.0 * 10
7～1.0 * 10
0Copy/μ L adds to the PCR reaction system by the template add-on in step 4, carries out pcr amplification according to step 4 method, with the template bottom line of determining that the method detects.
(b) stability and replica test
Respectively with foot and mouth disease virus O types 6 concentration, 10 times of serial dilutions, A type and Asia 1 type pGEM-T easy restructuring equivalent plasmid mixture (every kind of plasmid final concentration 1.0 * 10
7～1.0 * 10
2) carry out pcr amplification according to step 4 method, each series is set 3 repetitions, to verify the stable and repeated of the method.
(c) application test
According to foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type set up, the preparation detection reagent, foot and mouth disease virus O type, A type, the Asia 1 positive contrast of type type strain cell culture equal amount of mixture with deactivation, the normal negative contrast of BHK-21 cell, by the laboratory of domestic foot and mouth disease virus separation and Culture research qualification to 10 parts of clinical signs of suspected samples of its preservation (sample number into spectrum is: S1-S10) detect, and with its before isolation identification result relatively.
Description of drawings
Fig. 1: foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR product of type electrophorogram; Descriptive name: M. 100bp gradient DNA molecular weight marker, P1. foot and mouth disease virus O type, A type, Asia 1 type cell culture equal amount of mixture positive control, P2. foot and mouth disease O type culture positive control, P3. foot and mouth disease A type culture positive control, P4. foot and mouth disease Asia 1 type culture positive control, the N. negative control.
Fig. 2: foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR specific tests of type; Descriptive name: M.100bp gradient DNA molecular weight marker, 1. positive control, 2. negative control, 3. swine vesicular disease virus, 4. pig breeds and breathing syndrome virus, 5. pig vesicular stomatitis, 6. Pestivirus suis, 7. PRV (Pseudorabies virus), 8. streptococcus suis 2-type, 9. toxoplasma gondii.
Fig. 3: foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR sensitivity tests of type; Descriptive name: gradient DNA molecular weight marker M.100bp, 1～8 is three serotypes of foot and mouth disease virus (O type, A type, Asia 1 type) restructuring positive plasmid equal amount of mixture (1.0 * 10 of 10 times of serial dilutions
7～1.0 * 10
0Copy/μ L).
Fig. 4: foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR application tests of type; Descriptive name: gradient DNA molecular weight marker M.100bp, P: positive control, N: negative control, 1. S1,2. S2,3. S3,4. S4,5. S5,6. S6,7. S7,8. S8,9. S9,10. S10.
Embodiment
Materials and methods
1.1 material
1.1.1 strain
Through the foot and mouth disease virus O of deactivation type, A type and Asia 1 type type strain cell culture, from the foot and mouth disease detection kit of domestic certain production of units; Other contrast viruses or bacterial strain are preserved by Henan Province animal epidemic prevention and control centralab.
Instrument and reagent
The pcr amplification instrument, German Biometra company product; The gel imaging analysis system, U.S. Alpha Innotech company product; Thermostatic water bath vibrator (HZQ-Q), Harbin east connection electronic technology development corporation, Ltd. product; Table-type high-speed refrigerated centrifuge, U.S. Heraeus company product;
ExTaqArchaeal dna polymerase, dNTPs, DNA recovery test kit etc. are all available from Dalian (treasured) biotechnology company limited, and pGEM-T Easy carrier, JM109 competent cell are available from Promega company.
Method
1.2.1 design of primers and synthetic
Foot and mouth disease virus O type, A type and Asia 1 type complete genome sequence in a large amount of comparison GenBank, the 2B gene of selecting foot and mouth disease virus O type, A type and 1 three serotype high conservatives of Asia is that stencil design shares reverse transcription primer P0, according to these 3 serotype type specificity VP1 gene orders, design trizygoid Auele Specific Primer (P1/P2, P3/P4, P5/P6), synthetic by Dalian (treasured) biotechnology company limited, being diluted to final concentration is 20 μ mol/ L.Be used for foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR primer sequences of type as follows:
P0：tgtcctcctg catctggttg 20
P
1：gtccagagac gccaacacac g 21
P
2：ggtgttgtcc aacgctgtct cg 22
P
3：agccccacgc acgtcattga c 21
P
4：acccgcgccg cgagagacc 19
P
5：agcccaagag cacccaaacc c 21
P
6：acggcggtct tgtgtggtgt c 21
1.2.2 the preparation of template ribonucleic acid
Single and the positive contrast of equal amount of mixture through the foot and mouth disease virus O of deactivation type, A type and Asia 1 type type strain cell culture, the normal negative contrast of BHK-21 cell adopt the Trizol method to extract total RNA with detected sample simultaneously.concrete operations are as follows: get respectively inactivation of viruses cell culture monoculture and equal amount of mixture, each 200 μ L of negative control and sample to be checked (getting supernatant after appropriate PBS fully grinds as first adding when organizing) are in the 1.5mL centrifuge tube, add again the Trizol of 600 μ L to shake 2-3min in vortice, add 200 μ L chloroforms, 4 ℃, after the centrifugal 10min of 12000rpm, getting supernatant changes in another 1.5mL centrifuge tube, add 200 μ L isopropanol precipitatings, the washing with alcohol precipitation of mass percent 75%, dry, at last with 20 μ L DEPC water dissolution precipitations, get 10 μ L and be used for reverse transcription, all the other-20 ℃ of preservations.
Reverse transcription
Every pipe reverse transcription reaction system contains following composition: 5 * M-MLV Reaction buffer, 4 μ L; 2.5mmol/L dNTPs 4 μ L; M-MLV ThermoScript II 0.5 μ L; RNA enzyme inhibitors 0.5 μ L; P
0Primer 1 μ L, cumulative volume 10 μ L.The RNA 10 μ L that add respectively step 2 to make in every pipe reverse transcription reaction system, 37 ℃ of water-bath 1h or be placed in 37 ℃, PCR instrument reaction 1h, after reaction finishes, 70 ℃, 15 min deactivation ThermoScript II, be directly used in following pcr amplification or-20 ℃ frozen standby.
Pcr amplification and evaluation
Carry out the triple PCR amplification take reverse transcription product as template.By the optimization to PCR reaction system and reaction conditions, determine that reaction system is as follows: adopt 25 μ L reaction systems, wherein: 10 *
ExTaqBuffer 2.5 μ L, 2.5mM dNTPs 2 μ L,
The ExTaq polymerizationEnzyme 2.5 U, each 0.5 μ L of 3 pairs of type specificity primers (P1/P2, P3/P4, P5/P6), step
1.2.3The single cell culture of prepared O type, A type and Asia 1 type and equal amount of mixture, normal each 2 μ L of BHK-21 cell reverse transcription product mend to 25 μ L with deionized water, put on the PCR instrument and react.Amplification condition is 94 ℃, after 5min, and 94 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations, last 72 ℃ are extended 10min.The PCR product is put gel imaging analysis systematic observation result through 1.5 % agarose gel electrophoresis.
The product fragment of foot and mouth disease virus O type, A type and the positive amplification of Asia 1 type type strain is cloned into respectively pGEM-T easy carrier, screens positive recombinant plasmid and send Dalian precious biotechnology company limited to adopt T7 and SP6 primer to check order.
Specific test
By the 1.2.2 method to extraction RNA such as swine vesicular disease virus, pig breeding and breathing syndrome virus, pig vesicular stomatitis virus, Pestivirus suis and carry out reverse transcription by 1.2.3, PRV (Pseudorabies virus), streptococcus suis 2-type and toxoplasma gondii etc. directly extract DNA, establish simultaneously through the foot and mouth disease virus O of deactivation type, A type and the Asia 1 positive contrast of type type strain equivalent cell culture mixture, the normal negative contrast of BHK-21 cell cDNA is carried out pcr amplification with the specificity of checking the method according to the 1.2.4 method.
Sensitivity test
The foot and mouth disease virus O type, A type and the Asia 1 type pGEM-T easy recombinant plasmid that obtain in 1.2.4 are made respectively 10 times of serial dilutions, and three kinds of plasmid 1:1:1 ratios are mixed, and every kind of plasmid final concentration is adjusted into 1.0 * 10
7～1.0 * 10
0Copy/μ L adds to the PCR reaction system by the template add-on in 1.2.4, carries out pcr amplification according to the 1.2.4 method, with the template bottom line of determining that the method detects.
Stability and replica test
Respectively with foot and mouth disease virus O types 6 concentration, 10 times of serial dilutions, A type and Asia 1 type pGEM-T easy recombinant plasmid equal amount of mixture (every kind of plasmid final concentration 1.0 * 10
7～1.0 * 10
2) carry out pcr amplification according to the 1.2.4 method, each series is set 3 times and is repeated, to verify the stable and repeated of the method.
Application test
According to foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type set up, the preparation detection reagent, foot and mouth disease virus O type, A type, the Asia 1 positive contrast of type type strain cell culture equal amount of mixture with deactivation, the normal negative contrast of BHK-21 cell, by domestic have the laboratory of foot and mouth disease virus separation and Culture research qualification to 10 parts of clinical signs of suspected samples of its preservation (sample number into spectrum is: S1-S10) detect, and with its before isolation identification result relatively.
Result
2.1 the foundation of foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR diagnostic methods of type
Amplified simultaneously the fragment of three entries of 230bp, the 330bp of expection and 470bp left and right size through the foot and mouth disease virus O of deactivation type, A type and Asia 1 type type strain equivalent cell culture mixture, single strain cell culture has all amplified corresponding single band; Negative control does not amplify any band; Judge the detected result establishment; At this moment, if test sample the specific amplification band occurs in the position consistent with positive amplified band molecular size range, be judged to be the detected result positive, otherwise be judged to feminine gender.
To order-checking after the pcr amplification product clone of above three different sizes, the definite length of the pcr amplification product of O type, A type and Asia 1 type is respectively 234bp, 326bp and 467bp.Sequence is inputted carried out BLAST with listed foot-and-mouth disease virus gene sequence in GenBank and compare, three fragment gene sequences and O type, A type and Asia 1 type VP1 gene order homology are all more than 98%.
Specific test
Adopt the multiplex RT-PCR method of setting up that O type, A type and Asia 1 type type strain cDNA mixture are increased and to obtain the band that size is three entries of 234bp, 326bp and 467bp, but normal BHK-21 cell, swine vesicular disease virus, pig breeding and breathing syndrome virus, pig vesicular stomatitis, Pestivirus suis, PRV (Pseudorabies virus), streptococcus suis 2-type, toxoplasma gondii all fail to amplify any band.
Sensitivity test
Triple RT-PCR methods are 100 copy/μ L to foot and mouth disease virus O type, A type and the Asia 1 minimum detection limit of type recombinant plasmid, size occurring is the purpose band of 234bp, 326bp and 467bp, shows that it is 100 copy/μ L that foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type set up can detect bottom line.
Stability and replica test
The replica test result shows, concentration is 1.0 * 10
7～1.0 * 10
2The foot and mouth disease virus O type of 6 concentration of copy/μ L, A type and 3 revision test results of Asia 1 type recombinant plasmid equal amount of mixture are all positive, and concentration is 1.0 * 10
1Copy/μ L and 1.0 * 10
03 revision test results of copy/μ L recombinant plasmid equal amount of mixture are all negative.Illustrate that foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection methods of type set up have good stability and repeatability.
Application test
10 parts of clinical signs of suspected samples are carried out triple RT-PCR amplifications, 4 parts of foot and mouth disease virus O type nucleic acid positive, 1 part of foot and mouth disease virus A type nucleic acid positive, foot and mouth disease Asia 13 parts of type nucleic acid positive, 2 parts of negative samples as a result.This detected result with before the isolation identification and the sequencing result coincidence rate that have carried out be 100%.
Sequence table
＜110〉Henan Provincial Center for Animal Disease Control and Prevention
＜120〉foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection reagent of type and detection method thereof
〈160〉9
〈210〉1
〈211〉234
〈212〉DNA
＜213〉aphthovirus genus (Aphthovirus)
〈220〉
＜223〉foot and mouth disease virus O type VP1 gene order
〈400〉1
gtccagagac gccaacacac ggatgtctcg ttcatattag acagatttgt gaaagtaaca 60
ccaaaagacc aaattaatgt gttggacctg atgcaaaccc ctgcacacac tttggtaggc 120
gcgctcctcc gcactgccac ttactacttc gcagatttag aagtggcagt gaaacacgag 180
gggaacctta cctgggtccc gaacggggcg cccgagacag cgttggacaa cacc 234
〈210〉2
〈211〉326
〈212〉DNA
＜213〉aphthovirus genus (Aphthovirus)
〈220〉
＜223〉foot and mouth disease virus A type VP1 gene order
〈400〉2
agccccacgc acgtcattga cctcatgcaa acacaccaac acgcgttggt gggtgccctt 60
ttgcgtgcag ccacgtacta cttctccgat ctggagattg tggtgcgtca tgatggcaac 120
ttgacgtggg tgcccaatgg agcacctgta gaagccttgg ccaacacaag caaccccacc 180
gcctaccaca agcagccatt tacgagactt gcgctccctt acaccgcgcc gcaccgagtg 240
ttggcaacag tgtataacgg agtaagcagg tactctacaa ctggtaatgg cagaaggggt 300
gacctggggt ctcttgcggc gcgggt 326
〈210〉3
〈211〉467
〈212〉DNA
＜213〉aphthovirus genus (Aphthovirus)
〈220〉
＜223〉foot and mouth disease virus Asia 1 type VP1 gene order
〈400〉3
agcccaagag cacccaaacc cttgatctca tgcagatccc ctcacacacg ctggtcgggg 60
cgcttctccg gtctgcgacg tactacttct cagacctgga ggttgcgctc gtccacacag 120
gaccggtcac gtgggtgccc aatggtgcgc ccaagaccgc cttgaacaac cacaccaacc 180
cgactgccta ccagaagcag cctatcaccc gcttggcact cccctacacc gctccccacc 240
gtgtgttgtc aacagtgtac aacgggaaga caacgtacgg agaagaatcc tcgcggcgtg 300
gtgatcttgc cgcccttgca cgcagagtga acaaccggct gcccacttcc ttcaactacg 360
gcgctgtgaa ggccgacacc atcacggagc tgttgatccg catgaagcgt gcggaaacat 420
actgccccag gcccttgctg gctcttgaca ccacacaaga ccgccgt 467
〈210〉4
〈211〉20
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P0 primer is foot and mouth disease virus O type, A type, common reverse transcription primer used when the Asia 1 triple RT-PCR of type detect
〈400〉4
tgtcctcctg catctggttg 20
〈210〉5
〈211〉21
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P1 primer is amplification foot and mouth disease virus O type upstream primer used.
〈400〉5
gtccagagac gccaacacac g 21
〈210〉6
〈211〉22
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P2 primer is amplification foot and mouth disease virus O type downstream primer used.
〈400〉6
ggtgttgtcc aacgctgtct cg 22
〈210〉7
〈211〉21
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P3 primer is amplification foot and mouth disease virus A type upstream primer used
〈400〉7
agccccacgc acgtcattga c 21
〈210〉8
〈211〉19
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P4 primer is amplification foot and mouth disease virus A type downstream primer used
〈400〉8
acccgcgccg cgagagacc 19
〈210〉9
〈211〉21
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P5 primer is amplification foot and mouth disease virus Asia 1 type upstream primer used
〈400〉9
agcccaagag cacccaaacc c 21
〈210〉9
〈211〉21
〈212〉DNA
＜213〉synthetic
〈220〉
＜223〉P6 primer is amplification foot and mouth disease virus Asia 1 type downstream primer used
〈400〉9
acggcggtct tgtgtggtgt c 21
Claims (2)
1. a foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection reagent of type, it is characterized in that, comprise 1 shared reverse transcription primer P0, for O type, A type and 3 couples of type specificity primer P1/P2 of Asia 1 type, P3/P4, P5/P6, amplification target fragment length is respectively 234bp, 326bp and 467bp; 7 primed DNA sequences used are:
Primer P0:tgtcctcctg catctggttg 20
Primer P1:gtccagagac gccaacacac g 21
Primer P2:ggtgttgtcc aacgctgtct cg 22
Primer P3:agccccacgc acgtcattga c 21
Primer P4:acccgcgccg cgagagacc 19
Primer P5:agcccaagag cacccaaacc c 21
Primer P6:acggcggtct tgtgtggtgt c21.
2. application rights requires the non-diagnosis detecting method of 1 described foot and mouth disease virus O type, A type and the Asia 1 triple RT-PCR detection reagent of type, it is characterized in that, comprises the following steps:
⑴ the preparation of total RNA
with foot and mouth disease virus O type, single and the positive contrast of equal amount of mixture of A type and Asia 1 type cell deactivation poison type strain cell culture, the normal negative contrast of BHK-21 cell, adopt simultaneously the Trizol method to extract total RNA with detected sample, concrete operations are as follows: get respectively inactivation of viruses cell culture monoculture and equal amount of mixture, each 200 μ L of negative control and sample to be checked are in the 1.5ml centrifuge tube, add again the Trizol of 600 μ L to shake 2-3min in vortice, add 200 μ L chloroforms, 4 ℃, after the centrifugal 10min of 12000rpm, getting supernatant changes in another 1.5ml centrifuge tube, add 200 μ L isopropanol precipitatings, the washing with alcohol precipitation of mass percent 75%, dry, at last with 20 μ L DEPC water dissolution precipitations, get 10 μ L and be used for reverse transcription, all the other-20 ℃ of preservations,
⑵ reverse transcription
Every pipe reverse transcription reaction system contains following composition: 5 * M-MLV Reaction buffer, 4 μ L; 2.5mmol/L dNTPs 4 μ L; M-MLV ThermoScript II 0.5 μ L; RNA enzyme inhibitors 0.5 μ L; P
0Primer 1 μ L, cumulative volume 10 μ L; The RNA 10 μ L that add respectively step (1) gained in every pipe reverse transcription reaction system, 37 ℃ of water-bath 1h or be placed in 37 ℃, PCR instrument reaction 1h, after reaction finishes, 70 ℃, 15 min deactivation ThermoScript II, be directly used in following pcr amplification or-20 ℃ frozen standby;
⑶ pcr amplification and evaluation
Carry out the triple PCR amplification take reverse transcription product as template, by the optimization to PCR reaction system and reaction conditions, determine that reaction system is as follows: adopt 25 μ L reaction systems, wherein: 10 *
Ex TaqBuffer 2.5 μ L, 2.5mM dNTPs 2 μ L,
Ex Taq polymerizationEnzyme 2.5 U, article 6, each 0.5 μ L of primer, each 2 μ L of the reverse transcription product of O type, A type and Asia 1 type mend to 25 μ L with deionized water, put and carry out amplified reaction on the PCR instrument, amplification condition is 94 ℃, after 5min, and 94 ℃ of 45s, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations, last 72 ℃ are extended 10min.
⑷ result is judged
Amplified simultaneously the fragment of three entries of 234bp, the 326bp of expection and 467bp size through the foot and mouth disease virus O of deactivation type, A type and Asia 1 type type strain equivalent cell culture mixture, single strain cell culture has amplified corresponding single band; Negative control does not amplify any band, judges the detected result establishment; At this moment, if test sample the specific amplification band occurs in the position consistent with positive amplified band molecular size range, be judged to be the detected result positive, otherwise be judged to feminine gender.
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