CN104745729A - Double fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses and porcine reproductive and respiratory syndrome viruses and preparation method and application thereof - Google Patents

Double fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses and porcine reproductive and respiratory syndrome viruses and preparation method and application thereof Download PDF

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CN104745729A
CN104745729A CN201510190439.XA CN201510190439A CN104745729A CN 104745729 A CN104745729 A CN 104745729A CN 201510190439 A CN201510190439 A CN 201510190439A CN 104745729 A CN104745729 A CN 104745729A
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respiratory syndrome
swine fever
porcine reproductive
african swine
nsp2
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董志珍
王建华
赵祥平
柴铭骏
肖妍
赵丹
王玉玲
张俊哲
陈本龙
陈小金
王乃福
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Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
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Abstract

The invention discloses a double fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses and porcine reproductive and respiratory syndrome viruses and a preparation method and application thereof. Two sets of specific primers and Taqman probes as well as positive controls specific to African swine fever virus CP530R genes and porcine reproductive and respiratory syndrome virus NSP2 genes respectively are designed and synthesized, and a rapid, easy and convenient double real-time fluorescent quantitative RT-PCR detection system with high specificity and high sensitivity is established by using the two sets of primers and probes, so that nucleic acids of the African swine fever viruses and the porcine reproductive and respiratory syndrome viruses can be detected synchronously from a detected sample within 3-4 hours in a rapid, accurate, specific, safe, easy and convenient way. The detection reagent can be applied to synchronous detection of the nucleic acids of trace African swine fever viruses and porcine reproductive and respiratory syndrome viruses in hogs and relevant samples thereof.

Description

Bifluorescence RT-PCR detection reagent of African swine fever virus and porcine reproductive and respiratory syndrome virus and preparation method thereof and purposes
Technical field
The invention belongs to biological technical field, bifluorescence RT-PCR detection reagent being specifically related to a kind of African swine fever virus and porcine reproductive and respiratory syndrome virus and preparation method thereof and purposes.
Background technology
African swine fever (african swine fever, ASF) be that the one of the pig caused by African swine fever virus (ASFV) is acute, hot, high degree in contact communicable disease, it is characterized by that the course of disease is short, case fatality rate is high, clinical symptom and pathological change be all similar to acute swine fever, the high heat of performance, dermohemia, miscarriage, oedema and internal organs are hemorrhage.It is is breathed by pig and reproductive syndrome virus causes pig a kind of high degree in contact sexually transmitted disease that is cardinal symptom with wean front piglet high mortality, bred pigs respiratory symptom and sow breeding difficulty that pig breathes with reproductive syndrome.Similar behavior is had clinically with reproductive syndrome because African swine fever and pig breathe, only be not easily distinguishable with clinical symptom, therefore the discriminating that clinical doubtful morbidity pig carries out African swine fever virus and porcine reproductive and respiratory syndrome virus is detected, etiology foundation can be provided for finding early and controlling these two kinds of animal epidemics.Multiple fluorescence quantitative RT-PCR method can detect multiple cause of disease nucleic acid simultaneously, has the advantage that multiple cause of disease just can be differentiated and detect to a RT-PCR reaction easy, rapidly.Fluorescence RT-PCR method and the publication of the single African swine fever virus of current detection and porcine reproductive and respiratory syndrome virus all have report, and bifluorescence RT-PCR detection reagent that simultaneously can detect African swine fever virus and porcine reproductive and respiratory syndrome virus and preparation method thereof and application, also rare report.
Summary of the invention
Technical problem to be solved by this invention is, bifluorescence RT-PCR detection reagent that a kind of African swine fever virus and porcine reproductive and respiratory syndrome virus are provided and preparation method thereof and purposes, be specifically related to a kind of respectively with the detection reagent that African swine fever virus CP530R and porcine reproductive and respiratory syndrome virus NSP2 develops for amplified target sequence, this detection reagent comprises two pairs of Auele Specific Primers, two specific marker probes and two positive controls.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is realized by following steps: the bifluorescence RT-PCR detection reagent of a kind of African swine fever virus and porcine reproductive and respiratory syndrome virus, comprise two pairs of Auele Specific Primers for detecting African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene respectively, two specific probes and two positive controls, amplification target length is respectively 73bp and 95bp, and primer and probe sequence are: African swine fever virus
Upstream primer: CP530R-F:5 '-GTGTCAATAATCCCTATCTTGCCA-3 ' SEQ ID NO.1
Downstream primer: CP530R-R:5 '-AACTCGGTGGGATGGTTGAG-3 ' SEQ ID NO.2
Probe: CP530R-P:5 '-FAM-TCCTTGCTGTCTTTCTTGTC-3 '-MGB SEQ ID NO.3
Positive control: ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAATCAGTTTCCTTGCTGTCTTTC TTGTCG
CTCAACCATCCCACCGAGTTTATTAAGGTACTGCCGCTTATAGAC； SEQ ID NO.4
Porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-NSP2-F:5 '-CTCACAGACGGAATATGAGGC-3 ' SEQ ID NO.9
Downstream primer: PRRSV-NSP2-R:5 '-CACTCAGGACTTCCTCAGCTTC-3 ' SEQ ID NO.10
Probe: PRRSV-NSP2-P:5 '-VIC-CACCATCGCAGAAC-MGB-3 ' SEQ ID NO.11
Positive control: GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCT AGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGGGGGCAAGAAGCTGAGGAAG TCCTGAGTGAAATCTC
SEQ ID NO.12。
The preparation method of the bifluorescence RT-PCR detection reagent of above-mentioned African swine fever virus and porcine reproductive and respiratory syndrome virus, comprises the following steps:
(1) select African swine fever virus CP530R gene order conservative fragments to be amplification and the target sequence preparing positive control, its nucleotide sequence as shown in SEQ ID NO.4, for:
ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAATCAGTTTCCTTGCTGTCTTTCTTGTCGCTCAACCATCCCACCGAGTTTATTAAGGTACTGCCGCTTATAGAC；
(2) select porcine reproductive and respiratory syndrome virus NSP2 gene order conservative fragments to be amplification and the target sequence preparing positive control, its nucleotide sequence as shown in SEQ ID NO.12, for:
GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGGGGGCAAGAAGCTGAGGAAGTCCTGAGTGAAATCTC；
(3) design 4 primers, with pcr amplification and the contrast of preparation African swine fever virus CP530R gene masculine, primer sequence SEQ ID NO.5-SEQ ID NO.8 is as follows:
CP530RF1：5’-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3’
CP530RR1：5’-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-3’
CP530RF2：5’-ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAGTCGGTCTCCTTGCTG-3’
CP530RR2：5’-GTCTATAAGCGGCAGTACCTTAATAACTCGGTGGGATGGTTGAGGCGA-3’
(4) design 4 primers, with pcr amplification and prepare porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast, primer sequence SEQ ID NO.13-SEQ ID NO.16, as follows:
PRRSV-NSP2-F1:5’-GGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGG-3’
PRRSV-NSP2-R1:5’-CCCCGCCTCCAGGATGCCCATGTTCTGCGATGGTGCTAGGGGGAAAGCCTCATATTCC-3’
PRRSV-NSP2-F2:5’-GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTA-3’
PRRSV-NSP2-R2:5’-GAGATTTCACTCAGGACTTCCTCAGCTTCTTGCCCCCCCGCCTCCAGGATGCCCATG-3’
(3) multipair primer and many probes are designed according to African swine fever virus CP530R gene masculine control sequence and porcine reproductive and respiratory syndrome virus NSP2 gene masculine control sequence, through a large amount of contrast shaker tests, determine the suitableeest primer pair and label probe combination:
African swine fever virus
Upstream primer: CP530R-F:5 '-GTGTCAATAATCCCTATCTTGCCA-3 ' SEQ ID NO.1
Downstream primer: CP530R-R:5 '-AACTCGGTGGGATGGTTGAG-3 ' SEQ ID NO.2
Probe: CP530R-P:5 '-FAM-TCCTTGCTGTCTTTCTTGTC-3 '-MGB SEQ ID NO.3
Porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-NSP2-F:5 '-CTCACAGACGGAATATGAGGC-3 ' SEQ ID NO.9
Downstream primer: PRRSV-NSP2-R:5 '-CACTCAGGACTTCCTCAGCTTC-3 ' SEQ ID NO.10
Probe: PRRSV-NSP2-P:5 '-VIC-CACCATCGCAGAAC-MGB-3 ' SEQ ID NO.11
(6) fluorescent reporter group detecting 5 ' end mark of the probe of African swine fever virus CP530R gene is FAM, and the non-fluorescence quenching group of 3 ' end mark is MGB; The fluorescent reporter group detecting 5 ' end mark of the probe of porcine reproductive and respiratory syndrome virus NSP2 gene is VIC, and the non-fluorescence quenching group of 3 ' end mark is MGB;
(7) preferred through a large amount of simultaneous tests and reaction conditions, determine specificity and the sensitivity of reaction conditions, primer and probe that bifluorescence RT-PCR is the suitableeest.
The suitableeest reaction conditions in described (5) is that the upstream and downstream primer working concentration detecting African swine fever virus M gene and swine influenza virus M gene is 15pmol/ μ L, the working concentration of probe is 5.0pmol/ μ L, all adds primer and the probe of 0.5 μ L in 25 μ L systems; The specificity of primer and probe makes it be different from other virus of pig source, and the sensitivity detecting African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene reaches 61 copies and 46 copies respectively.
The application of bifluorescence RT-PCR detection reagent in production standard test kit of above-mentioned African swine fever virus and porcine reproductive and respiratory syndrome virus.
The bifluorescence RT-PCR detection reagent of above-mentioned African swine fever virus and porcine reproductive and respiratory syndrome virus is in the purposes of drawing African swine fever virus and porcine reproductive and respiratory syndrome virus fluorescence quantitative RT-RCR examination criteria curve.
The invention has the beneficial effects as follows: (1) has special, responsive, feature accurately and rapidly, can be used for detecting the micro-African swine fever virus nucleic acid in a pig and correlated samples thereof and porcine reproductive and respiratory syndrome virus nucleic acid simultaneously.(2) the African swine fever virus CP530R gene in the present invention and porcine reproductive and respiratory syndrome virus NSP2 genetic contrast preparation method, neither relate to the use of African swine fever virus and porcine reproductive and respiratory syndrome virus and genomic nucleic acids thereof, also the African swine fever virus CP530R gene DNA fragment not needing synthetic longer and porcine reproductive and respiratory syndrome virus NSP2 gene RNA fragment, have easy, safe and economic advantage.
Accompanying drawing explanation
Fig. 1 is the fluorescence RT-PCR amplification curve that the suitableeest primer pair contrasts African swine fever virus CP530R gene masculine with label probe.
Fig. 2 is that the suitableeest primer pair and label probe are to the fluorescence of African swine fever virus CP530R gene special RT-PCR specific amplification curve.
Fig. 3 is to 6.1 × 10 8~ 6.1 × 10 0the fluorescence quantitative RT-RCR amplification kinetic curve of the African swine fever virus CP530R gene masculine contrast in copies/ μ L concentration range.
Fig. 4 is the fluorescence quantitative RT-RCR typical curve of African swine fever virus CP530R gene.
Fig. 5 is the fluorescence RT-PCR amplification curve that the suitableeest primer pair contrasts porcine reproductive and respiratory syndrome virus NSP2 gene masculine with label probe.
Fig. 6 is that the suitableeest primer pair and label probe are to the fluorescence RT-PCR specific amplification curve of porcine reproductive and respiratory syndrome virus NSP2 gene.
Fig. 7 is to 4.6 × 10 8~ 4.6 × 10 0the fluorescence quantitative RT-RCR amplification kinetic curve of the porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast in copies/ μ L concentration range.
Fig. 8 is the fluorescence quantitative RT-RCR typical curve of porcine reproductive and respiratory syndrome virus NSP2 gene.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Bifluorescence RT-PCR detection reagent of African swine fever virus of the present invention and porcine reproductive and respiratory syndrome virus and preparation method thereof and purposes, comprise the following steps.
First step is the positive control substance of preparation African swine fever virus CP530R gene, and process comprises:
(1) in GenBank database, BLAST comparison is carried out to CP530R gene order, select one section of conserved sequence to be used for CP530R gene masculine contrast preparation, as shown in SEQ ID NO.4.
(2) synthesize 4 primers based on SEQ ID NO.4 sequences Design, primer sequence is as follows:
CP530RF15’-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3’
CP530RR15’-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-3’
CP530RF2：5’-ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAGTCGGTCTCCTTGCTG-3’
CP530RR2：5’-GTCTATAAGCGGCAGTACCTTAATAACTCGGTGGGATGGTTGAGGCGA-3’；
(3) using two complementary primers as amplification template, other two are carried out standard PCR amplification with the partly overlapping primer of primer sequence being used as template as amplimer;
(4) recovery, purifying contain the pcr amplification product of SEQ ID NO.1 sequence, be connected, be converted into DH5 α Recombinant organism with PCR2.1 cloning vector, picking, extraction positive colony plasmid;
(5) carry out quality determination with analyser, become copy number by formula scales, obtain African swine fever virus CP530R gene masculine contrast (plasmid) of known copy Particle density.
Second step prepares the contrast of porcine reproductive and respiratory syndrome virus NSP2 gene masculine, and process comprises:
(1) in GenBank database, BLAST comparison is carried out to porcine reproductive and respiratory syndrome virus NSP2 gene order, select one section of conserved sequence to be used for M gene masculine contrast preparation, as shown in SEQ ID NO.8.
(2) synthesize 4 primers based on SEQ ID NO.4 sequences Design, primer sequence is as follows:
PRRSV-NSP2-F1:5’-GGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGG-3’
PRRSV-NSP2-R1:5’-CCCCGCCTCCAGGATGCCCATGTTCTGCGATGGTGCTAGGGGGAAAGCCTCATATTCC-3’
PRRSV-NSP2-F2:5’-GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTA-3’
PRRSV-NSP2-R2:5’-GAGATTTCACTCAGGACTTCCTCAGCTTCTTGCCCCCCCGCCTCCAGGATGCCCATG-3’；
(3) using two complementary primers as amplification template, other two are carried out standard PCR amplification with the partly overlapping primer of primer sequence being used as template as amplimer;
(4) recovery, purifying contain the pcr amplification product of SEQ ID NO.8 sequence, be connected, be converted into DH5 α Recombinant organism with PCR2.1 cloning vector, picking, extraction positive colony plasmid;
(5) positive colony plasmid Bam H I enzyme of above-mentioned purifying is cut, reclaim digestion products, with linearizing positive colony plasmid DNA for template, use DNA in-vitro transcription test kit to carry out in-vitro transcription, use dnase digestion DNA profiling, extract RNA.
(6) carry out quality determination with DNA/RNA analyser, become copy number by formula scales, obtain porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast (RNA) of known copy Particle density.
Third step is primer, the probe of design and synthesis and selective mechanisms African swine fever virus CP530R gene, and process comprises:
(1) based on sequence shown in SEQ ID NO.1, the multipair primer of design and synthesis and multiple label probe;
(2) the African swine fever virus CP530R gene masculine control plasmids of the 1000 times of dilutions prepared with first step, for template, adopt One Step PrimeScript tMthe reaction amplification condition of RT-PCR Kit (Perfect Real Time) reagent and recommendation, the reaction amplification condition of amplifing reagent and recommendation, at Light 480 fluorescent PCR instrument carry out shaker test to the multipair primer of designed synthesis and label probe;
(3) under the reaction amplification condition of identical Fluorescence PCR amplifing reagent and recommendation, there is the synthetic determination standard of minimum Ct value, most high fluorescent increased value (Δ Rn) and typical S type amplification curve, amplification curve is shown in Fig. 1 respectively, and the suitableeest primer and the label probe sequence of the detection African swine fever virus CP530R gene finally determined are respectively: upstream primer: CP530R-F:5 '-GTGTCAATAATCCCTATCTTGCCA-3 '
Downstream primer: CP530R-R:5 '-AACTCGGTGGGATGGTTGAG-3 '
Probe: CP530R-P:5 '-FAM-TCCTTGCTGTCTTTCTTGTC-3 '-MGB.
4th step is primer, the probe of design and synthesis and selective mechanisms porcine reproductive and respiratory syndrome virus NSP2 gene, and process comprises:
(1) based on sequence shown in SEQ ID NO.4, the multipair primer of design and synthesis and multiple label probe;
(2) positive control (RNA) of the porcine reproductive and respiratory syndrome virus NSP2 genes of the 1000 times of dilutions prepared with first step, for template, adopts One Step PrimeScript tMthe reaction amplification condition of RT-PCR Kit (Perfect Real Time) reagent and recommendation, at Light 480 fluorescent PCR instrument carry out shaker test to the multipair primer of designed synthesis and label probe;
(3) there is the synthetic determination standard of minimum Ct value, the highest fluorescence intensity increased value (Δ Rn) and typical S type amplification curve, amplification curve is shown in Fig. 5 respectively, and the suitableeest primer pair and the label probe sequence of the detection porcine reproductive and respiratory syndrome virus NSP2 gene finally determined are respectively:
Upstream primer: PRRSV-NSP2-F:5 '-CTCACAGACGGAATATGAGGC-3 ' SEQ ID NO.9
Downstream primer: PRRSV-NSP2-R:5 '-CACTCAGGACTTCCTCAGCTTC-3 ' SEQ ID NO.10
Probe: PRRSV-NSP2-P:5 '-VIC-CACCATCGCAGAAC-MGB-3 ' SEQ ID NO.11
5th step is bifluorescence RT-PCR reaction system and the amplification condition of optimum detection African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene, and process comprises:
(1) primer third step and the 4th step determined and label probe, be mixed with the working concentration of 10pmol/ μ L, 8pmol/ μ L, 6pmol/ μ L, 4pmol/ μ L, 2pmol/ μ L with sterilized water respectively, probe sterilized water be mixed with respectively the working concentration of 3.5pmol/ μ L, 3pmol/ μ L, 2.5pmol/ μ L, 2.0pmol/ μ L, 1.5pmol/ μ L;
(2) One Step PrimeScript is adopted tMthe reaction amplification condition of RT-PCR Kit (Perfect Real Time) reagent and recommendation, at Light the primer that 480 fluorescent PCR instrument combine different working concentration and label probe carry out fluorescence RT-PCR square formation shaker test;
(3) be synthetic determination foundation to obtain minimum Ct value, higher fluorescence intensity increased value (Δ Rn) and typical S type amplification curve, the working concentration of the detection African swine fever virus CP530R gene finally determined and the suitableeest upstream and downstream primer of porcine reproductive and respiratory syndrome virus NSP2 gene is 15pmol/ μ L, the working concentration of the suitableeest probe is 5pmol/ μ L, in 25 μ L systems, add 0.5 μ L primer and probe;
(4) under the suitableeest primer and label probe concentration and universal fluorescent RT-PCR reaction amplifing reagent condition, within the scope of 61 DEG C ~ 56 DEG C, carry out shaker test to annealing elongating temperature, the suitableeest fluorescent PCR amplification condition finally determined is: 42 DEG C of reaction 5min; 94 DEG C of denaturation 10s; 94 DEG C of sex change 10s, 60 DEG C extend 20s, totally 40 circulations.
6th step is the bifluorescence RT-PCR method of primer and the label probe determined based on third step, the 4th step and the 5th step, set up the fluorescence RT-PCR amplification typical curve of detection African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene respectively and determine lowest detectable limit, process comprises:
(1) carry out 10 times of serial dilutions to the contrast of African swine fever virus CP530R gene masculine, the optimal reaction system determined with the 5th step and amplification condition carry out fluorescence RT-PCR amplification, and the kinetic curve of fluorescence RT-PCR amplification is shown in Fig. 3 respectively;
(2) with the Ct value of amplification curve for X-coordinate, with the logarithm of the copy number concentration of CP530R gene masculine contrast for ordinate zou drawing standard curve, the typical curve regression equation obtained is Y=-3.4496X+38.102, relation conefficient (r) is 0.9992 (see Fig. 4), show that designed primer and probe amplification efficiency and combination rate are high, the reaction conditions optimized is suitable for, at least 61 DNA moleculars copied can be detected, can be used for the detection by quantitative of micro-African swine fever virus CP530R gene, there is very high detection sensitivity.
(3) 10 times of serial dilutions are carried out to the contrast of porcine reproductive and respiratory syndrome virus NSP2 gene masculine, the optimal reaction system determined with the 5th step and amplification condition carry out fluorescence RT-PCR amplification, and the kinetic curve of fluorescence RT-PCR amplification is shown in Fig. 7 respectively;
(4) with the Ct value of amplification curve for X-coordinate, with the logarithm of porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast copy number concentration for ordinate zou drawing standard curve, the typical curve regression equation obtained is Y=-3.4189X+37.574, relation conefficient (r) is 0.9997 (see Fig. 8), show that designed primer and probe amplification efficiency and combination rate are high, the reaction conditions optimized is suitable for, at least 46 RNA molecule copied can be detected, can be used for the detection by quantitative of porcine reproductive and respiratory syndrome virus NSP2 gene, there is very high detection sensitivity.
7th step carries out specific test to the bifluorescence RT-PCR method of the primer determined based on third step, the 4th step and the 5th step and label probe, and process comprises:
(1) for cell culture and the foot and mouth disease vaccine of Pestivirus suis, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, swine influenza virus, adopt TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 test kit to press process specifications and extract geneome RNA;
(2) for the cell culture of Pseudorabies virus, pig parvoviral and pig circular ring virus, TaKaRaMiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 test kit is adopted press process specifications extraction genomic dna.
(3) with based on third step, the bifluorescence RT-PCR method of the primer that the 4th step and the 5th step are determined and label probe, to Pestivirus suis, the geneome RNA of swine foot-and-mouth disease virus and swine influenza virus and Pseudorabies virus, pig parvoviral, the genomic dna of pig circular ring virus carries out fluorescence RT-PCR amplification, detected result display is based on third step, the bifluorescence RT-PCR method of the primer that the 4th step and the 5th step are determined and label probe has good specificity, non-specific amplification curve (seeing Fig. 2 and Fig. 6 respectively) is there is not with above-mentioned pig correlated virus nucleic acid.
8th step be determine to detect African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene in family pig and correlated samples thereof working method, process comprises:
(1) process man pig and correlated samples method thereof is set up;
(2) extract the method for the RNA/DNA of sample simultaneously;
(3) utilize the bifluorescence RT-PCR method of primer and the label probe determined based on third step, the 4th step and the 5th step, African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene test and result are carried out to these samples and judges.
The preparation of embodiment 1 African swine fever virus CP530R gene masculine contrast
1. the selection of reference genetic sequence
BLAST is carried out to African swine fever virus CP530R gene order (the GenBank number of including: KJ380911.1), choose the reference sequences of one section of conservative and sequence of suitable design fluorescence PCR primer and probe as preparation African swine fever virus CP530R gene masculine control plasmid, as shown in SEQ ID NO.4.
2. amplimer Design and synthesis
Based on above-mentioned sequence, design and synthesis 4 pcr amplification primers, wherein CP530RF1 and CP530RR1 sequence complete complementary is used as template, CP530RF2 and CP530RR2 is overlapping with CP530RF1 and CP530RR1 Sequence respectively, and the sequence of synthesis is as follows:
CP530RF15-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3
CP530RR1：5-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-3
CP530RF2：5-ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAGTCGGTCTCCTTGCTG-3
CP530RR2：5-GTCTATAAGCGGCAGTACCTTAATAACTCGGTGGGATGGTTGAGGCGA-3
3.PCR increases
In 50 μ L PCR amplification system, add 10 × Buffer successively (containing MgCl 2) 5 μ L, dNTPs (2.5mmol/L) 4 μ L, TaKaRa Taq polysaccharase (5.0U/ μ L) 0.2 μ L, CP530RF1 (1pmmol/ μ L) and CP530RR1 (1pmmol/ μ L) each 1 μ L, CP530RF2 (25pmmol/ μ L) and CP530RR2 (25pmmol/ μ L) each 1 μ L, supplies ddH 2o to 50 μ L.
Pcr amplification reaction Parameter Conditions is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 20s, totally 35 circulations; 72 DEG C extend 6min; 4 DEG C of insulation 1min.
The recovery of 4.PCR product, purifying
Get the agarose gel electrophoresis that PCR primer carries out 1.5%, cutting size is the object amplified band of 121bp, and the process specifications purifying reclaiming test kit according to a small amount of sepharose DNA reclaims target DNA fragment.
The connection of 5.PCR product and cloning vector, conversion
According to PCR2.1 cloning vector process specifications, the target DNA fragment of purifying is spent the night with PCR2.1 cloning vector under 16 DEG C of conditions and is connected, this connection product is mixed with DH5 α Recombinant organism, 42 DEG C of heat shock 90s, after ice bath 2min, add the 37 DEG C of joltings of 1mL LB substratum and cultivate 1h, then in AMP +lB flat board is coated with rear 37 DEG C of incubated overnight.
6.CP530R gene masculine contrasts selecting, identify and measuring of (plasmid)
Picking 10 single bacterium colonies, 37 DEG C of jolting incubated overnight in 3mL LB substratum, then extract test kit with mini-scale plasmid and press process specifications Isolation and purification cloned plasmids, insert the positive colony plasmid of PP62 conserved sequence with PCR method screening; Carry out quality determination with the positive colony plasmid of DNA/RNA content analyzer to purifying, and according to following formula, plasmid mass conversion become copy number:
Namely be prepared into African swine fever virus CP530R gene masculine by aforesaid method to contrast.
The preparation of embodiment 2 porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast (RNA)
1. the selection of reference genetic sequence
BLAST is carried out to porcine reproductive and respiratory syndrome virus NSP2 gene order (the GenBank number of including: JX087437), choose one section of conservative and sequence of suitable design fluorescence RT-PCR primer and probe as the reference sequences preparing porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast (RNA), as shown in SEQ ID NO.1.
2. amplimer Design and synthesis
Based on above-mentioned sequence, design and synthesis 4 pcr amplification primers, wherein PRRSV-NSP2-F1 and PRRSV-NSP2-R1 sequence complete complementary is used as template, PRRSV-NSP2-F2 and PRRSV-NSP2-R2 respectively with PRRSV-NSP2-F1 and
PRRSV-NSP2-R1 Sequence is overlapping, and the sequence of synthesis is as follows:
PRRSV-NSP2-F1:5’-GGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGG-3’
PRRSV-NSP2-R1:5’-CCCCGCCTCCAGGATGCCCATGTTCTGCGATGGTGCTAGGGGGAAAGCCTCATATTCC-3’
PRRSV-NSP2-F2:5’-GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTA-3’
PRRSV-NSP2-R2:5’-GAGATTTCACTCAGGACTTCCTCAGCTTCTTGCCCCCCCGCCTCCAGGATGCCCATG-3’；
3.PCR increases
In 50 μ L PCR amplification system, add 10 × Buffer successively (containing MgCl 2) 5 μ L, dNTPs (2.5mmol/L) 4 μ L, TaKaRa Taq polysaccharase (5.0U/ μ L) 0.2 μ L, PRRSV-NSP2-F1 (1pmmol/ μ L) and PRRSV-NSP2-R1 (1pmmol/ μ L) each 1 μ L, PRRSV-NSP2-F2 (25pmmol/ μ L) and PRRSV-NSP2-R2 (25pmmol/ μ L) each 1 μ L, supplies ddH 2o to 50 μ L.
Pcr amplification reaction Parameter Conditions is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 20s, totally 35 circulations; 72 DEG C extend 6min; 4 DEG C of insulation 1min.
The recovery of 4.PCR product, purifying
Get the agarose gel electrophoresis that PCR primer carries out 1.5%, cutting size is the object amplified band of 128bp, and the process specifications purifying reclaiming test kit according to a small amount of sepharose DNA reclaims target DNA fragment.
The connection of 5.PCR product and cloning vector, conversion
According to PCR2.1 cloning vector process specifications, the target DNA fragment of purifying is spent the night with PCR2.1 cloning vector under 16 DEG C of conditions and is connected, this connection product is mixed with DH5 α Recombinant organism, 42 DEG C of heat shock 90s, after ice bath 2min, add the 37 DEG C of joltings of 1mL LB substratum and cultivate 1h, then in AMP +lB flat board is coated with rear 37 DEG C of incubated overnight.
6. insert the selecting of porcine reproductive and respiratory syndrome virus NSP2 gene masculine plasmid, identify
Picking 10 single bacterium colonies, 37 DEG C of jolting incubated overnight in 3mL LB substratum, then extract test kit with mini-scale plasmid and press process specifications Isolation and purification cloned plasmids, insert the positive colony plasmid of PP62 conserved sequence with PCR method screening;
7. porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrasts preparation and the copy number mensuration of (RNA)
Positive colony plasmid Bam H I enzyme of above-mentioned purifying is cut, reclaim digestion products, with linearizing positive colony plasmid DNA for template, use DNA in-vitro transcription test kit RiboMAXTM Large Scale RNA Production SystemT7, in-vitro transcription is carried out according to process specifications, use dnase digestion DNA profiling, extract RNA, packing.Carry out quality determination with the positive colony plasmid of DNA/RNA nucleic acid content analyser to purifying, and according to following formula, plasmid mass conversion become copy number:
Namely be prepared into porcine reproductive and respiratory syndrome virus NSP2 gene masculine by aforesaid method and contrast (RNA).
The drafting of embodiment 3 African swine fever virus CP530R gene by fluorescence quantitative PCR examination criteria curve
The dilution of 1.CP530R gene masculine contrast (plasmid)
Be 6.1 × 10 to concentration 10african swine fever virus CP530R gene masculine contrast (plasmid) carry out 10 times of serial dilutions, select 6.1 × 10 8~ 6.1 × 10 1doubly CP530R gene masculine contrast (plasmid) of dilution is as standard substance template.
2. the system of fluorescence RT-PCR reaction is prepared and amplification
Adopt the One Step PrimeScript of the employing TaKaRa company of TaKaRa company tMrT-PCR Kit (Perfect Real Time) fluorescence RT-PCR reaction reagent, by the reaction system composition preparation reaction system of table 1, fully mix packing 24.0 μ L in backward each fluorescent PCR pipe, then CP530R gene masculine contrast (plasmid) of 1.0 μ L10 times serial dilutions is added successively, each extent of dilution does 3 Parallel testing systems, after centrifugal after sealing, be positioned over Light in pattern detection groove on 480 fluorescent PCR instrument.Reaction parameter is 42 DEG C of 5min, 95 DEG C of 10sec, then 95 DEG C of 10sec, 56 DEG C of 10sec, 60 DEG C of 30sec, 40 circulations, before the 60 DEG C of annealing that circulate each time terminate, gather FAM channel fluorescence signal.
Table 1 fluorescence RT-PCR reaction system forms
3. the drafting of typical curve
Reaction terminate after, African swine fever virus CP530R gene masculine contrast fluorescent PCR amplification kinetic curve see Fig. 3, African swine fever virus CP530R gene masculine contrast copy number concentration with corresponding Ct value in table 2.
The corresponding Ct value of each copy number of fluorescence quantitative RT-RCR of table 2 African swine fever virus CP530R gene masculine contrast
With the Ct value of amplification curve for X-coordinate, with the copy number concentration of African swine fever virus CP530R gene masculine contrast for ordinate zou drawing standard curve, the typical curve regression equation obtained is Y=-3.4867X+38.313, relation conefficient (r) is 0.9994 (see Fig. 4), show that designed primer and probe amplification efficiency and combination rate are high, the reaction conditions optimized is suitable for, at least 61 DNA moleculars copied can be detected, can be used for the detection by quantitative of micro-African swine fever virus CP530R gene, there is very high detection sensitivity.
The drafting of embodiment 4 porcine reproductive and respiratory syndrome virus NSP2 gene fluorescence quantitative RT-PCR examination criteria curve
1. the dilution of porcine reproductive and respiratory syndrome virus NSP2 gene (RNA)
Be 4.6 × 10 to concentration 9porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast (RNA) carry out 10 times of serial dilutions, select 4.6 × 10 8~ 4.6 × 10 1doubly porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast (RNA) of dilution is as standard substance template.
2. the system of fluorescence RT-PCR reaction is prepared and amplification
Adopt the One Step PrimeScript of TaKaRa company tMrT-PCR Kit (Perfect Real Time) fluorescence RT-PCR reaction reagent, by the reaction system composition preparation reaction system of table 3, fully mix packing 24.0 μ L in backward each fluorescent PCR pipe, then CP204L gene masculine contrast (plasmid) of 1.0 μ L10 times serial dilutions is added successively, each extent of dilution does 3 Parallel testing systems, after centrifugal after sealing, be positioned over Light in pattern detection groove on 480 fluorescent PCR instrument.Reaction parameter is 42 DEG C of 5min, 95 DEG C of 10sec, then 95 DEG C of 10sec, 56 DEG C of 10sec, 60 DEG C of 30sec, 40 circulations, before the 60 DEG C of annealing that circulate each time terminate, gather VIC channel fluorescence signal.
Table 3 fluorescence RT-PCR reaction system forms
3. the drafting of typical curve
Reaction terminate after, porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast fluorescence RT-PCR amplification kinetic curve see Fig. 7, porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast copy number concentration with corresponding Ct value in table 4.
The corresponding Ct value of each copy number of fluorescence quantitative RT-RCR of table 4 porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast
With the Ct value of amplification curve for X-coordinate, with the copy number concentration of porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast for ordinate zou drawing standard curve, the typical curve regression equation obtained is Y=-3.378X+37.545, relation conefficient (r) is 0.9975 (see Fig. 8), show that designed primer and probe amplification efficiency and combination rate are high, the reaction conditions optimized is suitable for, at least 46 RNA molecule copied can be detected, can be used for the detection by quantitative of micro-porcine reproductive and respiratory syndrome virus NSP2 gene, there is very high detection sensitivity.
The detection of African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene in embodiment 5 actual sample
1. the process of sample and genomic dna/RNA thereof extract
(1) process of sample
For pig blood blood sample, directly draw 200 μ L, extract for genomic dna/RNA; For pork sample, with aseptic scissors and tweezers clip measuring samples 2.0g in mortar, abundant grinding, then add 10mL PBS and mix, or be placed in tissue homogenizer, add 10mL PBS homogenate, then tissue suspension is proceeded in aseptic Eppendorf pipe, the centrifugal 10min of 3000r/min, get supernatant liquor and proceed in another aseptic 1.5mL centrifuge tube, draw 200 μ L suspensions, extract for genomic dna/RNA.
(2) genomic dna/RNA extracts
TaKaRa MiniBEST Viral RNA/DNAExtraction Kit Ver.5.0 test kit is adopted to press the genomic dna/RNA of process specifications extraction for pig blood sample and treated pork sample; Meanwhile, the genomic dna/RNA of positive control and negative control (normal negative porcine tissue sample) is processed and extracts by the same method.
2. the preparation of fluorescence RT-PCR reaction system and amplification condition
(1) reaction system preparation
Adopt the One Step PrimeScript of TaKaRa company tMrT-PCR Kit (Perfect Real Time) fluorescence RT-PCR reaction reagent, by the reaction system composition preparation reaction system of table 3, fully mix packing 16.5 μ L in backward each fluorescent PCR pipe, then 8.5 μ L detection of nucleic acids samples are added, after centrifugal after sealing, be positioned over Light in pattern detection groove on 480 fluorescent PCR instrument.
Table 3 bifluorescence RT-PCR reaction system forms
(2) amplification condition
Reaction parameter is 42 DEG C of 5min, 95 DEG C of 10sec, then 95 DEG C of 10sec, 56 DEG C of 10sec, 60 DEG C of 30sec, 40 circulations, gathers the fluorescent signal of FAM passage and VIC passage before the 60 DEG C of annealing that circulate each time terminate respectively.
3. result judges
After off-test, observe amplification curve and the Ct value of the positive control of FAM passage and VIC passage, negative control and sample, judge to test and whether set up and the result of pattern detection.When negative control is without Ct value and without amplification curve, the Ct value of positive control should≤30.0, and occur specific amplification curve, then test establishment, otherwise this time experiment be considered as invalid.Ct value≤30.0, and there is specific amplification curve, represent in sample to there is virus, be judged to positive findings, without Ct value and without amplification curve, show in sample virus-free, be judged to negative findings.
The present invention establishes a kind of fast and convenient, high specificity, highly sensitive bifluorescence RT-PCR detection system, African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene can be detected quick from tested sample, accurate, special, safe in 3 ~ 4 hours, easily, can be used for the detection from African swine fever virus CP530R gene micro-in pig blood sample and pork sample and porcine reproductive and respiratory syndrome virus NSP2 gene.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment all comprises within the scope of the present invention.

Claims (5)

1. the bifluorescence RT-PCR detection reagent of an African swine fever virus and porcine reproductive and respiratory syndrome virus, it is characterized in that, comprise two pairs of Auele Specific Primers for detecting African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene respectively, two specific probes and two positive controls, amplification target length is respectively 73bp and 95bp, and primer and probe sequence are:
African swine fever virus
Upstream primer: CP530R-F:5 '-GTGTCAATAATCCCTATCTTGCCA-3 ' SEQ ID NO.1
Downstream primer: CP530R-R:5 '-AACTCGGTGGGATGGTTGAG-3 ' SEQ ID NO.2
Probe: CP530R-P:5 '-FAM-TCCTTGCTGTCTTTCTTGTC-3 '-MGB SEQ ID NO.3
Positive control: ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAATCAGTTTCCTTGCTGTCTTTC TTGTCGCTCAACCATCCCACCGAGTTTATTAAGGTACTGCCGCTTATAGAC; SEQ ID NO.4
Porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-NSP2-F:5 '-CTCACAGACGGAATATGAGGC-3 ' SEQ ID NO.9
Downstream primer: PRRSV-NSP2-R:5 '-CACTCAGGACTTCCTCAGCTTC-3 ' SEQ ID NO.10
Probe: PRRSV-NSP2-P:5 '-VIC-CACCATCGCAGAAC-MGB-3 ' SEQ ID NO.11
Positive control: GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCT AGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGGGGGCAAGAAGCTGAGGAAG TCCTGAGTGAAATCTC
SEQ ID NO.12。
2. require the preparation method of the bifluorescence RT-PCR detection reagent of African swine fever virus as described in 1 and porcine reproductive and respiratory syndrome virus as profit, it is characterized in that, comprise the following steps:
(1) select African swine fever virus CP530R gene order conservative fragments to be amplification and the target sequence preparing positive control, its nucleotide sequence as shown in SEQ ID NO.4, for:
ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAATCAGTTTCCTTGCTGTCTTTCTTGTCGCTCAACCATCCCACCGAGTTTATTAAGGTACTGCCGCTTATAGAC；
(2) select porcine reproductive and respiratory syndrome virus NSP2 gene order conservative fragments to be amplification and the target sequence preparing positive control, its nucleotide sequence as shown in SEQ ID NO.12, for:
GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGGGGGCAAGAAGCTGAGGAAGTCCTGAGTGAAATCTC；
(3) design 4 primers, with pcr amplification and the contrast of preparation African swine fever virus CP530R gene masculine, primer sequence SEQ ID NO.5-SEQ ID NO.8 is as follows:
CP530RF1：5’-TCTTGCCAAGTCGGTCTCCTTGCTGTCTTTCTTGTCGCCTCAACCATCCCACCGAGTT-3’
CP530RR1：5’-AACTCGGTGGGATGGTTGAGGCGACAAGAAAGACAGCAAGGAGACCGACTTGGCAAGA-3’
CP530RF2：5’-ACTGGTGTAGTGTCAATAATCCCTATCTTGCCAAGTCGGTCTCCTTGCTG-3’
CP530RR2：5’-GTCTATAAGCGGCAGTACCTTAATAACTCGGTGGGATGGTTGAGGCGA-3’
(4) design 4 primers, with pcr amplification and prepare porcine reproductive and respiratory syndrome virus NSP2 gene masculine contrast, primer sequence SEQ ID NO.13-SEQ ID NO.16, as follows:
PRRSV-NSP2-F1:5’-GGAATATGAGGCTTTCCCCCTAGCACCATCGCAGAACATGGGCATCCTGGAGGCGGGG-3’
PRRSV-NSP2-R1:5’-CCCCGCCTCCAGGATGCCCATGTTCTGCGATGGTGCTAGGGGGAAAGCCTCATATTCC-3’
PRRSV-NSP2-F2:5’-GATGAGCCTCTGGATTTGTCTGCGTCCTCACAGACGGAATATGAGGCTTTCCCCCTA-3’
PRRSV-NSP2-R2:5’-GAGATTTCACTCAGGACTTCCTCAGCTTCTTGCCCCCCCGCCTCCAGGATGCCCATG-3’
(3) multipair primer and many probes are designed according to African swine fever virus CP530R gene masculine control sequence and porcine reproductive and respiratory syndrome virus NSP2 gene masculine control sequence, through a large amount of contrast shaker tests, determine the suitableeest primer pair and label probe combination:
African swine fever virus
Upstream primer: CP530R-F:5 '-GTGTCAATAATCCCTATCTTGCCA-3 ' SEQ ID NO.1
Downstream primer: CP530R-R:5 '-AACTCGGTGGGATGGTTGAG-3 ' SEQ ID NO.2
Probe: CP530R-P:5 '-FAM-TCCTTGCTGTCTTTCTTGTC-3 '-MGB SEQ ID NO.3
Porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-NSP2-F:5 '-CTCACAGACGGAATATGAGGC-3 ' SEQ ID NO.9
Downstream primer: PRRSV-NSP2-R:5 '-CACTCAGGACTTCCTCAGCTTC-3 ' SEQ ID NO.10
Probe: PRRSV-NSP2-P:5 '-VIC-CACCATCGCAGAAC-MGB-3 ' SEQ ID NO.11
(6) fluorescent reporter group detecting 5 ' end mark of the probe of African swine fever virus CP530R gene is FAM, and the non-fluorescence quenching group of 3 ' end mark is MGB; The fluorescent reporter group detecting 5 ' end mark of the probe of porcine reproductive and respiratory syndrome virus NSP2 gene is VIC, and the non-fluorescence quenching group of 3 ' end mark is MGB;
(7) preferred through a large amount of simultaneous tests and reaction conditions, determine specificity and the sensitivity of reaction conditions, primer and probe that bifluorescence RT-PCR is the suitableeest.
3. the preparation method of the bifluorescence RT-PCR detection reagent of African swine fever virus according to claim 2 and porcine reproductive and respiratory syndrome virus, it is characterized in that, the suitableeest reaction conditions in described (5) is that the upstream and downstream primer working concentration detecting African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene is 15pmol/ μ L, the working concentration of probe is 5.0pmol/ μ L, all adds primer and the probe of 0.5 μ L in 25 μ L systems; The specificity of primer and probe makes it be different from other virus of pig source, and the sensitivity detecting African swine fever virus CP530R gene and porcine reproductive and respiratory syndrome virus NSP2 gene reaches 61 copies and 46 copies respectively.
4. the application of bifluorescence RT-PCR detection reagent in production standard test kit of African swine fever virus as claimed in claim 1 and porcine reproductive and respiratory syndrome virus.
5. the bifluorescence RT-PCR detection reagent of African swine fever virus as claimed in claim 1 and porcine reproductive and respiratory syndrome virus is in the purposes of drawing African swine fever virus and porcine reproductive and respiratory syndrome virus fluorescence quantitative RT-RCR examination criteria curve.
CN201510190439.XA 2015-04-21 2015-04-21 Double fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses and porcine reproductive and respiratory syndrome viruses and preparation method and application thereof Pending CN104745729A (en)

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CN106834539A (en) * 2017-01-20 2017-06-13 北京市动物疫病预防控制中心 Highly pathogenic PRRSV and CSFV simultaneous quantitative detection kit
CN109825642A (en) * 2019-02-25 2019-05-31 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） For detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement
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CN105543410A (en) * 2015-12-25 2016-05-04 四川农业大学 Method for detecting pig viral diseases on basis of TEM-PCR and gene chip
CN105543410B (en) * 2015-12-25 2019-06-07 四川农业大学 Method based on TEM-PCR and genechip detection porcine viral diseases
CN106834539A (en) * 2017-01-20 2017-06-13 北京市动物疫病预防控制中心 Highly pathogenic PRRSV and CSFV simultaneous quantitative detection kit
CN109825642A (en) * 2019-02-25 2019-05-31 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） For detecting real-time fluorescence quantitative PCR primer, probe and its application of the related cyclic annular single-stranded DNA viruses of fur sea dog excrement
CN112779250A (en) * 2019-11-11 2021-05-11 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Kit for identifying porcine reproductive and respiratory syndrome virus and/or African swine fever virus and application thereof
CN112779352A (en) * 2019-11-11 2021-05-11 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Double digital PCR detection technology for swine fever and African swine fever and special kit thereof
CN112779250B (en) * 2019-11-11 2022-12-13 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Kit for identifying porcine reproductive and respiratory syndrome virus and/or African swine fever virus and application thereof
CN112779352B (en) * 2019-11-11 2023-07-21 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Dual digital PCR detection technology for swine fever and African swine fever and special kit thereof

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