基于P18蛋白基因的新型鸭呼肠孤病毒荧光定量RT-PCR检测方法的建立  

Establishment of SYBR Green I fluorescence quantitative RT-PCR assay for detection of novel duck reovirus based on the gene encoding P18 protein

作　　者：沈丽雅 潘群兴[1] 卢凤英[1] 董永毅 张敬峰[1] 吴坤 刘青涛[1] 姜平 张小飞[1] SHEN Li-ya;PAN Qun-xing;LU Feng-ying;DONG Yong-yi;ZHANG Jing-feng;WU Kun;LIU Qing-tao;JIANG Ping;ZHANG Xiao-fei(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Provincial Center for Animal Disease Control and Prevention,Nanjing 210009,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095 [3]江苏省动物疫病预防控制中心,江苏南京210009

出　　处：《江苏农业学报》2021年第6期1481-1487,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家重点研发计划项目(2017YFD0500804);江苏现代农业(水禽)产业技术体系疾病防控创新团队项目[JATS(2020)321]。

摘　　要：根据新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)特有的非结构蛋白P18基因保守序列设计1对特异性引物,建立了NDRV荧光定量RT-PCR检测方法。用该方法和普通RT-PCR方法对239份临床样品进行检测,NDRV阳性检出率分别为23.85%(57/239)和17.57%(42/239),荧光定量RT-PCR检测方法的检出率明显高于普通RT-PCR检测方法。随机取5份荧光定量RT-PCR检测方法检测的阳性的样品用鸭胚进行病毒分离培养,经普通RT-PCR扩增和基因测序证明均为NDRV。用10^(3.0) ELD_(50)剂量的NDRV-SY株人工感染14日龄雏鸭,不同时间采集泄殖腔肛拭子样品,通过荧光定量RT-PCR检测方法进行排毒情况监测,结果显示,雏鸭人工感染NDRV 48 h时可以从泄殖腔检测到病毒排出,持续时间可达20 d,并发现在病毒感染后的第5 d和第10 d出现2次排毒高峰。试验结果表明,建立的NDRV荧光定量RT-PCR检测方法具有良好的特异性和较高的敏感性,可用于NDRV感染的排毒监测以及临床感染的早期诊断和流行病学调查。In order to rapidly detect the novel duck reovirus(NDRV),the SYBR Green I fluorescence quantitative RT-PCR method was established with a pair of specific primers designed according to the conservative gene sequence of the unique non-structural protein P18 of NDRV.239 clinical samples were detected by the fluorescence quantitative RT-PCR method and ordinary RT-PCR methods,and the positive detection rates of the samples were 23.85%(57/239)and 17.57%(42/239),respectively.The detection rate of fluorescence quantitative RT-PCR method was significantly higher than that of ordinary RT-PCR method.Five samples were randomly selected for virus culture with duck embryo from the NDRV positive samples by the fluorescence quantitative RT-PCR method.It was proved that the five isolated viruses were NDRV by ordinary RT-PCR amplification and DNA sequencing technique.The 14-day-old ducklings were artificially infected with NDRV-SY strain at a dose of 10^(3.0) ELD_(50),and the cloaca swabs of these ducklings were collected for NDRV detection by this fluorescence quantitative RT-PCR at different time points.The results showed that the inoculated ducklings shed virus as early as 48 hours post inoculation,and could continue until 20 days post inoculation.In addition,two peaks of viral titers were observed at five and ten days post inoculation.Therefore,the established fluorescence quantitative RT-PCR is specific and sensitive,and can be used for the detection of NDRV,early diagnosis of clinical infection and epidemiological investigation.

关 键 词：新型鸭呼肠孤病毒 P18蛋白基因 荧光定量RT-PCR 

分 类 号：S858.325.3[农业科学—临床兽医学]