凡纳滨对虾肠道上皮细胞微胞子虫PCR检测方法的建立与应用

Development and Application of a PCR Detection Assay of Microsporidium Enterocytozoon hepatopenaei in Pacific White Leg Shrimp Litopenaeus vannamei

根据Gen Bank中对虾肠道上皮细胞微胞子虫(Enterocytozoon hepatopenaei,EHP)的基因保守序列,设计一对特异性引物,通过优化PCR扩增条件,建立快速检测凡纳滨对虾(Litopenaeus vannamei)EHP的PCR方法。用该方法对EHP阳性虾进行PCR扩增,得到与实验设计相符的330 bp特异性扩增条带,而对白斑综合症病毒(WSSV)、桃拉病毒(TSV)、传染性皮下及造血器官坏死病毒(IHHNV)、对虾杆状病毒(BP)、"棉花虾"微孢子虫、黏孢子虫的阳性虾,以及健康虾的扩增结果为阴性。测序比对发现,PCR产物序列与Gen Bank EHP基因序列的同源性为99.8%,表明该PCR方法检测结果准确。敏感性试验表明,该方法最低可检测出约100 fg的EHP质粒DNA。用该PCR方法检测广东、广西、江苏、山东、海南等地的755份临床样品,共检出阳性样品21份。该PCR方法可用于凡纳滨对虾EHP的快速检测。

A pair of specific primers was designed, and a rapid PCR method was established fordetection of microsporidium Enterocytozoon hepatopenaei in Pacific white leg shrimp Litopenaeusvannamei according to the published gene sequences in GenBank by optimization of the reactionparameters. The specific band of 330 bp were amplified from the positive shrimps, but no specificband was found from shrimps with white spot syndrome virus (WSSV), Taura syndrome virus (TSV),infectious hypodermal and hematopoietic necrosis virus (IHHNV), Baculovirus penaei (BP), and Pleistophora-like microsporidium, Myxosporea and healthy shrimps. According to the Sequencinganalysis, the sequence of PCR product had 99.8% homology with that of EHP gene in GenBankwhich indicated the high accuracy of this method. The sensitivity test of this method showed that thelowest detectable limit DNA plasmid of the microsporida was 100 fg. 755 clinical shrimp samplescollected from provinces of Guangdong, Guangxi, Jiangsu, Shandong, and Hainan were detected bythis method, and then 21 positive samples were found. The findings indicated that the PCR assaycould be used in the detection of Pacific white leg shrimp samples.