【摘要】 近年来，虾肝肠胞虫（Enterocytozoon hepatopenaei,EHP）流行区域不断扩大，已经成为我国南美白对虾（Litopenaeus vannamei）养殖重要病原之一。本研究根据Gen Bank中虾肝肠胞虫18S r RNA保守区域设计一套特异性引物和荧光探针，通过优化反应条件，建立检测EHP的TaqMan探针实时荧光定量PCR(Real-time quantitative polymerase chain reaction,qPCR)检测方法。建立的方法呈良好的线性关系（R~2=0.998），灵敏度是巢式PCR的10倍。特异性试验表明：检测其他常见病原均为阴性，特异性好；重复性试验显示：批内批间变异系数均小于1.5%，重复性良好；对临床样品检测，与巢式PCR的符合率为97.81%。使用该方法对中山市、江门市和珠海市珠三角部分地区南美白对虾开展EHP流行病学调查，其中虾苗检出率为10.75%，成虾检出率为54.07%，部分区域检出率较高，要防范爆发风险。本研究建立的实时荧光定量PCR灵敏度高、特异性和重复性良好，具有较好的应用前景。更多还原

【Abstract】 In recent years, the epidemic area of parasite Enterocytozoon hepatopenaei(EHP)has been spread, and become one of the important pathogens in the culture of Pacific white shrimp Litopenaeus vannamei in China. In this study, a set of specific primers and fluorescent probes were designed according to the conserved region of EHP 18s rRNA in GenBank. A real-time quantitative polymerase chain reaction method for detection of EHP with TaqMan probe was established by optimization of the reaction conditions, the test of the established method showed excellent linearity(R~2= 0.998)and 10 times higher sensitivity than nested PCR. Specificity tests showed that the established method had negative results for other common pathogens, with good specificity. The repeatability test showed that the intra assay interassay coefficients of variation were all less than 1.5%, with good repeatability. In clinical samples tested, the established method showed the concordance of 97.81% with nested PCR. This method was used to carry out EHP epidemiological investigation on Pacific white shrimp in some areas in the Pearl River Delta. The detection rates of Pacific white shrimp were found to be 10.75% in seedling and 54.07% in adult shrimp, with so high level of the detection rate in some regions that the outbreak risk should be prevented. The qPCR established in this study has high sensitivity, specificity and reproducibility, thus a good application prospect.更多还原