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草鱼呼肠孤病毒(Grass carp reovirus,GCRV)为草鱼出血病的病原.本实验根据GenBank中GCRV和其他水生呼肠孤病毒毒株的第六基因片段,在其保守区设计了一对GCRV特异性引物,建立了快速检测GCRV的逆转录聚合酶链式反应(RT-PCR)方法.该PCR体系中,上下游引物的最适终浓度为120 nmol/L,最适退火温度为52℃.PCR特异性试验表明:所设计的引物只能扩增GCRV的核酸,而不能扩增嗜水气单胞菌BSK-10、WSSV以及正常CIK细胞的DNA或RNA.敏感性试验表明,当GCRV的反转录模板稀释至5-7时,PCR结果还为阳性.用所建立的RT-PCR方法对5份样品进行检测,结果表明本研究建立的RT-PCR检测方法可靠且可行.
Grass carp reovirus (GCRV) is the pathogen of grass carp hemorrhage.According to the sixth gene fragment of GCRV and other hydatidous reovirus strains in GenBank, a pair of GCRV specific A rapid reverse transcription polymerase chain reaction (RT-PCR) method was established for the detection of GCRV.The optimal final concentration of the primers was 120 nmol / L and the optimal annealing temperature was 52 ℃. PCR specificity test showed that the designed primers can only amplify GCRV nucleic acid, but not DNA or RNA of Aeromonas hydrophila BSK-10, WSSV and normal CIK cells.A sensitivity test showed that when GCRV PCR results were also positive when the reverse transcription template was diluted to 5-7, and 5 samples were detected by RT-PCR. The results showed that the RT-PCR assay established in this study was reliable and feasible.