虾苗场3种重要病原同步定量PCR检测技术的建立  

A synchronous quantitative PCR assay for simultaneous detection of three significant pathogens in shrimp hatcheries

作　　者：李楠英 房文红[1] 王元[1] 马清扬 李新苍[1] 赵姝[1] 符贵红[1] 周俊芳[1] LI Nanying;FANG Wenhong;WANG Yuan;MA Qingyang;LI Xincang;ZHAO Shu;FU Guihong;ZHOU Junfang(Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation and Utilization,Ministry of Agriculture and Rural Affairs,East China sea Fisheries Research Institute,China Academy of Fishery Sciences,Shanghai 200090,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院东海水产研究所,农业农村部东海渔业资源开发利用重点实验室,上海200090 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋渔业》2020年第3期375-384,共10页Marine Fisheries

基　　金：中国水产科学研究院基本科研业务费(2017HY-ZD0302);上海市虾类产业技术体系建设项目[沪农科产字(2014)第5号];公益性行业(农业)科研专项基金(201303047)。

摘　　要：白斑综合征病毒(white spot syndrome virus,WSSV)、虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)和虾虹彩病毒(decapod iridescent virus 1,DIV1)为近年来凡纳滨对虾(Litopenaeus vannamei)育苗场内的主要生物性危害因素,其中2种或3种病原共感染的现象也较为常见,快速、准确地检测和鉴别这3种病原是大型苗场生物安保体系构建和无特定病原虾苗生产的迫切需求。基于SYBR Green I染料法,建立了同步扩增3种病原的定量PCR检测技术。结果显示,该同步方法对3种病原的标准曲线相关系数(R 2)均大于0.99,且检测限可低至10 copy·μL^-1,尤其是WSSV,在低至1 copy·μL^-1时仍能保持较好的组内和组间重复性。熔解曲线分析显示,该同步定量PCR技术对对虾样品的扩增产物只在78.4℃、79.7℃、83.5℃处出现3个尖锐峰,分别与3种病原(DIV1、EHP和WSSV)扩增产物的T m值相对应。以上结果表明,建立的同步定量PCR检测技术具有较高的检测灵敏度和良好的病原特异性,可以在50 min内同步完成对3种病原的快速鉴定和组织内病原含量的确定,既可用于对虾育苗场内对这3种病原的监测,也适用于对养殖对虾病原感染与病害发生的风险预测。White spot syndrome virus(WSSV),Enterocytozoon hepatopenaei(EHP)and decapod iridescent virus 1(DIV1)have been main biological hazards in Litopenaeus vannamei hatcheries in recent years,and co-infections with two or three pathogens are common.Rapid and accurate detection and identification of these three pathogens is an urgent need for the construction of biosecurity system and the production of specific pathogen free(SPF)seed in large-scale shrimp hatcheries.Based on the SYBR Green I,a quantitative PCR technique for simultaneous detection of the three pathogens was developed.Correlation coefficients(R 2)for all three pathogens were greater than 0.99 with the detection limits as low as 10 copy·μL^-1.Especially for WSSV,when the detection limit was as low as 1 copy·μL^-1,the technique still maintained good levels of intra-group and inter-group repeatabilities.Melting curve analysis showed that there were only three sharp peaks of the amplified products of shrimp samples at 78.4℃,79.7℃,83.5℃,which corresponded to the T m values of DIV1,EHP and WSSV,respectively.In contrast,no melting peaks were observed in all of the negative controls.The above results showed that the synchronous quantitative PCR(qPCR)assay had both high detection sensitivity and good pathogen specificity,which could quickly identify the three pathogens and determine the pathogen content in tissues within 50 min.Therefore,the synchronous qPCR assay can be used not only to monitor the three pathogens in shrimp hatcheries,but also to predict the risk of pathogenic infection and disease occurrence in shrimp farms.

关 键 词：白斑综合征病毒 虾肝肠胞虫 虾虹彩病毒 同步定量PCR 

分 类 号：S945.1[农业科学—水产养殖]