鲤疱疹病毒2型微滴式数字PCR快速检测方法的建立  

Establishment of droplet digital PCR assay for the rapid detection of cyprinid herpesvirus 2

作　　者：郝中香[1] 林华[2] 佘容 廖红[1] 陈世界[2] 杨苗[2] 薛昌华[2] 赵珊[1] 韩国全[4] 颜其贵[1] 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川出入境检验检疫局技术中心,四川成都610041 [3]通威股份有限责任公司检测中心,四川成都610041 [4]四川农业大学食品学院,四川雅安625014

出　　处：《中国兽医科学》2016年第2期167-173,共7页Chinese Veterinary Science

基　　金：四川出入境检验检疫局项目(SK201414;SK201411;SK201403)

摘　　要：根据GenBank上登录的鲤疱疹病毒2型(cyprinid herpesvirus 2,Cy HV-2)ORF6基因序列(登录号:JQ815364.1)用Primer Express 3.0设计并合成ORF6基因特异性引物及Taq Man探针,建立了检测鲤疱疹病毒2型的微滴式数字PCR(dd PCR)方法。结果显示,dd PCR的检测灵敏度比qPCR高5倍。特异性和重复性检测结果表明,ddPCR和qPCR均具有良好的特异性和较高的重复性。qPCR和dd PCR标准曲线的r2值分别为0.999 2和0.997 5,表明ddPCR和qPCR均具有良好的线性关系。研究结果表明,该方法可以对CyHV-2进行可靠的检测,可以满足CyHV-2特性研究和感染诊断的需要。对60份临床样品的检测结果显示,在检出阳性的34份样品中,包含肾、脾、腮、肝、心和脑,表明该病毒在这些器官均有分布,其中肾的病毒含量相对最高,脑的病毒含量相对最低。总之,本研究建立的ddPCR方法灵敏度高、特异性强、能快速检测到极低含量的病毒,适用于CyHV-2的快速检测和病原流行病学调查。The primers and probe were designed for cyprinid herpesvirus 2（Cy HV-2）qPCR and droplet digital PCR（dd PCR）by using Primer Express 3.0 Analysis Software based on the nucleotide sequences of ORF6 retrieved from Gen Bank（JQ815364.1）. The result showed that the sensitivity for dd PCR assay was advantageous over qPCR, which was 5 times higher than that of the qPCR. The specificity and reproducibility test showed that both dd PCR and q PCR methods had advantages of specificity and reproducibility. The r2 value of q PCR and dd PCR standard curve was 0.999 2 and 0.997 5, respectively, showed dd PCR and qPCR had a good linear response. These results indicated that the method can be reliable detection for CyHV-2, and could be satisfied for the studies on characteristics of CyHV-2 and the diagnosis needs of infecting CyHV-2. Sixty clinical samples were detected and the results showed that the kidney, spleen, gill, liver, heart and brain were positive, indicated that the virus was distributed in these organs.The q PCR results showed that the relative level was the highest in kidney and was lowest in brain.In conclusion,the established assay in this study had high sensitivity and specificity for rapid detecting low levels of CyHV-2,and can be applied in rapid detection and epidemiological survey of CyHV-2 infection in fish.

关 键 词：鲤疱疹病毒2型 微滴式数字PCR 检测 

分 类 号：S852.659.1[农业科学—基础兽医学]