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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
刘 珍,张庆利,万晓媛,马 芳,黄 倢
作者单位
刘 珍 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 
张庆利 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 
万晓媛 中国水产科学研究院黄海水产研究所 青岛 266071 
马 芳 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 
黄 倢 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 
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/µl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8.3×101 copies/µl,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 I
DOI:10.11758/yykxjz.20150512003
分类号:
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
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,HUANG Jie
Abstract:
In this study we developed fluorescence SYBR Green I real-time quantitative PCR (qPCR) to detect shrimp microsporidian Enterocytozoon hepatopenaei (EHP). A pair of specific primers was designed according to the SSU rDNA sequences of shrimp EHP published in GenBank and the optimized annealing temperature was determined to be 60℃. The melting curve of amplified products exhibited only one specific peak. Between 8.3×101–8.3×108 copies/µl the test results were linearly correlated with the titers of EHP. The cycles of amplification threshold (Ct) and the logarithmic of the initial template quantity [log(Sq)] conformed to Ct = –3.369 log(Sq) + 39.364, of which the coefficient of association R2 was 0.992 and the amplification efficiency was 98.1%. This method had high sensitivity (8.3×101 copies/µl), and generated duplicable results both within a group and between different groups. The test of 31 samples of farmed L. vannamei suggested that the qPCR method was 4 times more sensitive than the previously reported nested PCR method. To further verify this method, we tested hepatopancreatic DNA (HpDNA) samples extracted from 94 samples of L. vannamei that were collected from farms in Jiangsu, Hainan, and Shandong provinces. The results showed that the EHP loads in the hepatopancreas were negatively correlated with the shrimp growth rates. EHP load above 103 copies/(ng HpDNA) indicated high risk. In conclusion, we developed a highly specific, sensitive, rapid and quantitative method, which could be a useful tool in the prevention and control of shrimp microsporidian E. hepatopenaei.
Key words:  Real-time quantitative PCR  Microsporidian Enterocytozoon hepatopenaei  SYBR Green I