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

Abstract:

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.