RT-PCR检测草鱼呼肠孤病毒的方法研究-《集美大学学报（自然科学版）》

文章信息/Info

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

作者:: 郝贵杰¹; 2; 盛鹏程¹; 林锋¹; 潘晓艺¹; 徐洋¹; 姚嘉赟¹; 尹文林¹; 曹铮¹; 袁雪梅¹; 蔺凌云¹; 沈锦玉¹; 浙江省淡水水产研究所,浙江湖州 313001 ;2.宁波大学海洋学院,浙江宁波 315211 )

Author(s):: HAO Gui-jie¹; 2; SHENG Peng-cheng¹; LIN Feng¹; PAN Xiao-yi¹; XU Yang¹; YAO Jia-yun¹; YIN Wen-lin¹; CAO Zheng¹; YUAN Xue-mei¹; LIN Ling-yun¹; SHEN Jin-yu¹; (1.Zhejiang Institute of Freshwater Fisheries,Huzhou 313001, China;2.College of Marine,Ningbo University,Ningbo 315211, China)

Keywords:: GCRV; Ctenopharyngodon idellus kidney cell ; reverse transcription polymerase chain reaction; cytopathic effct; rapid diagnosis

摘要:: 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

Abstract:: 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.