RT—PCR检测草鱼呼肠孤病毒的方法研究

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120nmol／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．

Development and Application of RT-PCR Technique for Detecting Grass Carp Reovirus(GCRV) Infection

Grass carp reovirus （GCRV） is an important pathogenic virus, which can cause outbreak of grass carp hemorrhage in China freshwater region. A pair of specific primers was designed to amplify partial segment （about 440bp） of the sixth gene according to the sixth gene sequence of GCRV and other Aquareoviruses strains in GenBank. The reaction conditions of the RT - PCR were optimized and the optimum primers concentration was 120nmol/L of each and the optimum annealing temperature was 52℃. The primer specificity was evaluated and the results showed that the expected 440bp fragment could be amplified only from GCRV but not from Aeromonas hydrophila BSK - 10, WSSV, or uninfected CIK cells. The result of sensitivity showed that the positive 440 fragment could be amplified from the cDNA template even if the template diluted to 5 -7 times. Five samples of visceral organs of the moribund grass carp with hemorrhage symptom were detected using the RT-PCR method developed above, and the results showed that it was a useful method for detecting the pathogen of GCRV in diagnosis practice.