虾类行动障碍野田村病毒(MDNV) TaqMan RT-PCR检测方法的建立与应用

Establishment and application of TaqMan RT-PCR detection method for shrimp movement disorder nodavirus (MDNV)

中文关键词: 养殖对虾  行动障碍野田村病毒(MDNV)  TaqMan RT-PCR  定量  检测方法

英文关键词: Farming shrimp  Movement disorder nodavirus (MDNV)  TaqMan RT-PCR  Quantification  Detection method

中文摘要:

      基于虾类行动障碍野田村病毒(movement disorder nodavirus, MDNV)的RNA依赖的RNA聚合酶(RdRp)基因设计寡核苷酸引物和探针，以含有MDNV靶基因的pMD18-MDNV质粒和RNA标准品为模板，通过优化反应体系和反应程序，建立了MDNV的TaqMan RT-PCR检测方法。优化后的反应组分：20.0 μL TaqMan RT-PCR反应液预混液中包含11.0 μL一步法RT-PCR缓冲液、0.8 μL酶混合物、0.3 μmol/L正向引物、0.3 μmol/L反向引物、0.4 μmol/L探针、1.0 μL模板和5.2 μL RNA-free H2O；优化后的反应程序：54.5℃ 15 min，95℃ 1 min；45个热循环扩增(95℃ 10 s，60.3℃ 30 s)。本研究所建立的方法能实现对MDNV的特异性检测，在1.4×1010~1.4×101 copies/μL标准质粒浓度范围内，起始模板浓度对数值与反应循环数间呈良好的线性关系；该方法最低可检测5.5×101拷贝的RNA标准品或1.4×101拷贝的pMD18-MDNV标准质粒。分析结果还显示，该检测方法批内Ct值和批间Ct值的变异系数(CV)分别小于1.27%和1.83%，具有良好的重复性和稳定性。利用该方法对2019年采自我国部分省市的样品进行检测发现，凡纳滨对虾(Litopenaeus vannamei)中MDNV的阳性检出率为23.5% (16/68)。本研究建立的MDNV TaqMan RT-PCR方法具有特异性、快速和灵敏等特点，可为对虾养殖实践中MDNV的定性、定量检测与监测以及有效防控提供技术支持。

英文摘要:

      For the establishment of the TaqMan RT-PCR detection method for the shrimp movement disorder nodavirus (MDNV), primers and probes were designed based on the RNA-dependent RNA polymerases (RdRp). The pMD18-MDNV plasmid and RNA standard containing the MDNV target gene were used as templates to optimize the reaction mixtures and program. The optimized reaction with 20.0 μL master mix included the following components: 11.0 μL one-step RT-PCR buffer, 0.8 μL enzyme mixture, 0.3 μmol/L forward primer, 0.3 μmol/L reverse primer, 0.4 μmol/L probe, 1.0 μL template, and 5.2 μL RNA-free H2O. The optimized reaction procedure was as following: incubation at 54.5℃ for 15 min, incubation at 95℃ for 1 min, then 45 thermal cycling amplifications (95℃ for 10 s, 60.3℃ for 30 s). The newly established method was specific for MDNV detection, showing a good linear relationship between the log value (Starting quality, Sq) and number of reaction cycles within the range of 1.4×1010~1.4×101 copies/μL pMD18-MDNV standard plasmid. The method could detect as low as 5.5×101 copies of the RNA standard or 1.4×101 copies of the pMD18-MDNV standard plasmid. Meanwhile, the newly developed assay showed that the coefficient of variation of the Ct value intra-assay and the Ct value inter-assay were less than 1.27% and 1.83%, respectively, indicating a good repeatability and stability. In the samples collected from shrimp farming provinces in China in 2019 using this new method, the positive detection rate of MDNV in Litopenaeus vannamei was 23.5% (16/68). The TaqMan RT-PCR method established in this study was specific, fast, and sensitive in the detection of MDNV. This method could provide technical support for the qualitative and quantitative detection and monitoring of MDNV in shrimp farming practices, as well as effective prevention and control of MDNV.