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虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测
引用本文:刘 珍,张庆利,万晓媛,马 芳,黄 倢.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J].海洋水产研究,2016,37(2):119-126.
作者姓名:刘 珍  张庆利  万晓媛  马 芳  黄 倢
作者单位:1. 中国水产科学研究院黄海水产研究所 青岛266071;上海海洋大学水产与生命学院 上海201306;2. 中国水产科学研究院黄海水产研究所 青岛266071;青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266237;3. 中国水产科学研究院黄海水产研究所 青岛266071
基金项目:公益性行业科研专项经费项目(201103034)、现代农业产业技术体系(CARS-47)、山东省泰山学者建设工程专项经费和农业部科研杰出人才和创新团队专项经费共同资助
摘    要:根据GenBank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei) (EHP) SSU rDNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green Ⅰ实时荧光定量PCR (qPCR)检测方法.结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101-8.3×108 copies/μ1的EHP SSU rDNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数log(Sq)]的关系为Ct=-3.369 log(Sq)+39.364 (R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3× 10(1) copies/μ1,在线性范围内具有良好的组内和组间重复性.对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍.利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA (HpDNA)中的EHP SSU rDNA进行了qPCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng HpDNA)时代表了较高的风险水平.本研究建立的qPCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考.

关 键 词:实时荧光定量PCR  虾肝肠胞虫  SYBR  Green  
收稿时间:2015/5/12 0:00:00
修稿时间:2015/6/12 0:00:00

Development of Real-Time PCR Assay for Detecting Microsporidian Enterocytozoon hepatopenaei and the Application in Shrimp Samples with Different Growth Rates
LIU Zhen,ZHANG Qingli,WAN Xiaoyuan,MA Fang and HUANG Jie.Development of Real-Time PCR Assay for Detecting Microsporidian Enterocytozoon hepatopenaei and the Application in Shrimp Samples with Different Growth Rates[J].Marine Fisheries Research,2016,37(2):119-126.
Authors:LIU Zhen  ZHANG Qingli  WAN Xiaoyuan  MA Fang and HUANG Jie
Institution:Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;College of Fishery and Life Sciences, Shanghai Ocean University, Shanghai 201306;,Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200,Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;,Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071; College of Fishery and Life Sciences, Shanghai Ocean University, Shanghai 201306; and Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200
Abstract:
Keywords:Real-time quantitative PCR  Microsporidian Enterocytozoon hepatopenaei  SYBR Green I
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