张 娜，谢艳辉，陈进会，李家侨，斯泽恩，黄 磊

（1. 湛江出入境检验检疫局，广东湛江 524000；2. 东莞出入境检验检疫局，广东东莞 441900）

虾肝肠胞虫Taqman荧光PCR方法的建立与应用

张 娜1，谢艳辉1，陈进会2，李家侨1，斯泽恩1，黄 磊1

（1. 湛江出入境检验检疫局，广东湛江 524000；2. 东莞出入境检验检疫局，广东东莞 441900）

为给虾肝肠胞虫（EHP）的监测和控制工作提供特异、灵敏的快速检测方法，针对EHP 18S相关保守序列设计1对特异性引物，优化并建立EHP的Taqman荧光PCR检测方法，同时验证该方法的重复性和敏感性。结果显示：体系扩增的最佳退火温度为60 ℃，构建好的质粒标准品标准曲线斜率为-3.195，R2为0.991；扩增产物阈值循环数（Ct）与标准模板起始量（X）的关系曲线为-3.195×Log（X）+36.923，扩增效率为105.59。使用所构建方法检测湛江市EHP流行情况，同时对样品进行白斑综合症病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）、桃拉综合征病毒（TSV）、早期死亡综合症（AHPND）病毒检测。结果显示：样品的WSSV、IHHNV、TSV检测均为阴性；AHPND检出阳性率为10%，EHP为30%，其中1份样品的EHP和AHPND检测同为阳性。本研究建立的EHP荧光PCR方法具有特异、灵敏等优点。该方法及其临床应用数据可为EHP的防控提供技术参考。

虾肝肠胞虫；南美白对虾；Taqman荧光PCR

Abstract：To provide specific，sensitive and rapid detection methods for the monitoring and control of Enterocytozoon hepatopenaei（EHP），Taqman fluorescent PCR method to detect EHP of Shrimp microsporidian was developed，a pair of specific primers was designed according to the conserved sequences of EHP 18S published in Genbank，its repeatability and sensitivity were detected in this study. The results showed that the optimum annealing temperature was 60 ℃，the standard cycles of amplification threshold（Ct）and the logarithmic of the initial template quantity[log（Sq）] conformed to Ct=-3.195×Log（X）+36.923，of which the coefficient of association R2was 0.991，the slope was -3.195，the amplification efficiency was 105.59. The results showed that the method had nice property and high sensitivity. By examing the growth of the 20 vannamei samples collected from Zhanjiang area with the method of this experiment，the samples were also examined for WSSV，IHHNV，TSV and AHPND，test results showed that WSSV，IHHNV and TSV had incidence of 0，AHPND had incidence of 10%，EHP had incidence of 30%，and one shrimp sample showed co-infection of AHPND and EHP. In conclusion，a highly specific，sensitive method was developed，which could be a useful tool in the prevention and control of Shrimp microsporidian E.hepatopenaei.

Key words：Enterocytozoon hepatopenaei；Shrimp microsporidian；Taqman fluorescent PCR method

随着南美白对虾养殖规模的不断扩大，疫病成为对虾养殖业发展的瓶颈之一。虾急性肝胰腺坏死/虾早死综合症（AHPND/EMS）[1]、虾肝肠胞虫病[2]等新发疫病的发生，更加重了养殖企业的风险和负担，使得我国对虾养殖成功率逐年下降，严重影响了对虾进出口贸易和对虾养殖产业的发展。据湛江出入境检验检疫局的数据统计，近几年湛江市对虾出口不断下滑，2015年出口对虾14.95万吨，同比减少16.28%。……