Dual real-time fluorescent RT-PCR identification and detection method of PRRSV classic strain and highly pathogenic mutant strain
A PRRSV-R3, highly pathogenic technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of time-consuming, labor-intensive, co-infection, low sensitivity, etc., and improve detection efficiency , Reduce detection cost, good reliability
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Embodiment 1
[0037] Construction of standard RNA
[0038] (1) Experimental reagents
[0039] The restriction enzyme Pst I, the corresponding buffer 10×H Buffer and DL2000Marker were purchased from TaKaRa Company;
[0040] -T Easy ligation kit, Taq DNA polymerase, AMV reverse transcriptase, RNase inhibitor, dNTPs were purchased from Promega;
[0041] DNA gel recovery kit, purchased from OMEGA company;
[0042] High-efficiency Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;
[0043] Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.;
[0044] Other biochemical reagents are imported subpackages or domestic analytical pure.
[0045] (2) Experimental equipment
[0046] TGRANDIENT PCR instrument: purchased from HYBAID company;
[0047] DYFIII2 electrophoresis instrument: purchased from Bio-Rad;
[0048] UVP gel imaging analysis system: purchased from Gene Company;
[0049] Steril Biological saf...
Embodiment 2
[0064] Extraction of sample RNA
[0065] According to the manufacturer's recommendations, according to the routine method, using QIAGEN Mini Kit kit (purchased from QIAGEN), or according to the GTC method (guanidine isothiocyanate method) ( The Total RNA Isolation System kit was purchased from Promega) to extract RNA from samples to be tested from various sources.
Embodiment 3
[0067] Screening test of specific primers and probes for classic PRRSV strains and highly pathogenic variant strains by real-time fluorescent RT-PCR
[0068] (1) Experimental reagents
[0069] One Step PrimeScript TM RT-PCR Kit (Perfect Real Time) kit was purchased from TaKaRa Company;
[0070] The TaqMan MGB probes and primers for classic strains of PRRSV and highly pathogenic variants were synthesized by Shanghai Jikang Biotechnology Co., Ltd.
[0071] (2) Experimental equipment
[0072] Applied Biosystems 7500 Real-Time PCR System
[0073] (3) Experimental process
[0074] ① Design of primers and probes for classic PRRSV strains and highly pathogenic variant strains
[0075] The specific primers for classic PRRSV strains and highly pathogenic variant strains refer to: oligonucleotide chains with a length of about 20 bases, which are highly conserved with the ORF1a gene of classic PRRSV strains and highly pathogenic variant strains The specific nucleotide fragments ar...
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