Variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof

[0103] The mutant porcine reproductive and respiratory syndrome virus TaqMan fluorescent quantitative RT-PCR detection kit of the present invention. It includes the following components: DEPC water, 5×AMV buffer, 2.5mM / L dNTP, 2.5umol / LMgcl 2 , 10xPCR Buffer, Nuclease Inhibitor, AMV reverse transcriptase, LA TaqDNA polymerase, Oligo(dT)18, 100nmol / L primer mixture, 10nmol / LTaqMan probe, positive plasmid standard, negative control (physiological saline),

[0104] (1) Upstream and downstream primer mixtures: According to the nsp2 sequence of PRRSV-VR2332, PRRSV-FJ04a, PRRSV-JXwn06 (EF641008.1) mutants provided by GenBank database, the plasmid was designed and constructed with PrimerExpress5.0 software and Oligo6.0 software A pair of primers and probes, UP Primer: 5'-AGCTGATGACACCTTTGAGT-3'LOW Primer: 5'-AGCCTCATATTCCGTCTGTG-3', the target fragment size is 125bp,

[0105] (2)TaqMan probe:

[0106] Fluorescent probe sequence 5’FAM-CCGCGTAGAACTGTGACAACAACGCT-TAMRA 3’

[0107] (3) Positive...

[0120] The application of the mutant porcine reproductive and respiratory syndrome virus TaqMan fluorescent quantitative RT-PCR detection kit of the present invention: (1). The DEPC water in the kit, 5×AMV buffer, 10×PCR Buffer, 2.5mmol / L dNTP, 2.5umol / L Mgcl 2 ,, Oligo(dT)18, 100nmol / L primer mixture, 10nmol / LTaqMan probe, dissolve at room temperature;

[0121] (2) Synthesize the primer mixture and probe,

[0122] (3) Establish a fluorescence quantitative PCR reaction system,

[0123] Fluorescence quantitative RT-PCR reaction system 25ul, 10xPCR Buffer 13.5ul, 2.5mM / L dNTP2.5ul, 100nmol / L upstream and downstream primers 0.8ul each, 10nmol / L probe 0.8ul, LA TaqDNA polymerase 2U, template 1.0ul , The rest is sterilized distilled water to make up to 25μl, the reaction conditions are 94℃ pre-denaturation 5min, then 94℃10s, 55℃20s, 70℃20s, a total of 45 cycles,

[0124] (4)Mg 2+ , Primer and probe concentration optimization

[0125] Take the plasmid standard as the test sample, and the Mg...