虾肝肠胞虫Taqman荧光PCR方法的建立与应用

Establishment and Application of Taqman Fluorescent PCR Method for Detection of Enterocytozoon hepatopenaei

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。

To provide specific,sensitive and rapid detection methods for the monitoring and control of Enterocytozoonhepatopenaei(EHP),Taqman fluorescent PCR method to detect EHP of Shrimp microsporidian was developed,apair of specific primers was designed according to the conserved sequences of EHP18S published in Genbank,itsrepeatability and sensitivity were detected in this study.The results showed that the optimum annealing temperaturewas60℃,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 R2was0.991,the slopewas-3.195,the amplification efficiency was105.59.The results showed that the method had nice property and highsensitivity.By examing the growth of the20vannamei samples collected from Zhanjiang area with the method of thisexperiment,the samples were also examined for WSSV,IHHNV,TSV and AHPND,test results showed thatWSSV,IHHNV and TSV had incidence of0,AHPND had incidence of10%,EHP had incidence of30%,andone shrimp sample showed co-infection of AHPND and EHP.In conclusion,a highly specific,sensitive method wasdeveloped,which could be a useful tool in the prevention and control of Shrimp microsporidian E.hepatopenaei.