Development and application of duplex fluorescent quantitative PCR method for detecting Macrobrachium rosenbergii nodavirus and decapod iridescent virus 1

【Objective】To develop duplex fluorescent quantitative PCR detection method for Macrobrachium rosenbergii nodavirus（MrNV）and decapod iridescent virus（DIV1），so as to provide a simpler and more effective detection technology for clinical diagnosis and epidemic monitoring of M. rosenbergii white tail disease（WTD）and decapod iridescent virus 1 disease（DIV1D）.【Method】Specific primers and TaqMan-Minor Groove Binder（TaqMan-MGB）probes were designed based on the conserved sequence of MrNV-CP gene and DIV1-MCP gene respectively，and the target gene fragments were cloned into the pGM-T carrier to construct recombinant plasmids，pGM-T-CP^MrNV and pGM-T-MCP^DIV1. Among them，pGM-T-CP^MrNV was digested by SalⅠand transcribed in vitro to obtain MrNV standard RNA，while DNA of pGMT-MCP^DIV1 were served as DIV1 standard DNA. The reaction conditions were optimized with standard being taken as templates. And the standard curves were established. Then the duplex fluorescent quantitative PCR detection method for MrNV and DIV1 was developed，and the specificity，sensitivity，reproducibility tests and detection of clinical samples were carried out to evaluate the applicability of this method.【Result】The prepared RNA transcribed and DNA had good stability and could meet the requirements of the standards，and the standard curves showed a good linear relationship with quantitative range from 1×10¹ to 1×10⁸ copies/reaction. The optimized duplex fluorescent quantitative PCR method had a high sensitivity with the detection limit as low as to 10 copies/reaction for the standards and approximately 10 copies/mg for the M. rosenbergii tissue samples. It had no cross reaction with common shrimp pathogens，and the entire detection could be completed within 1 h for a single sample；the variation coefficients of intra-group and inter-group were 0.31%-0.93% and 0.96%-1.33% for MrNV detection，which were 0.35%-0.81% and 0.78%-1.04% for DIV1 detection. The duplex fluorescent quantitative PCR method and nested PCR method recommended by current standards were adopted to detect 117 clinical samples，the results showed that most of the detection result were the same with coincidence rate of 99.15% for MrNV detection and 97.44% for detecting DIV1. But the duplex fluorescent quantitative PCR method had better sensitivity in detecting samples with low copies target virus. The MrNV and DIV1 positive rates of Guangxi were 6.0% and 11.0% in 2020 respectively.【Conclusion】The duplex fluorescent quantitative PCR method developed based on TaqMan-MGB probe technology can detect MrNV and DIV1 virus at the same time and it is sensitive，specific，reproducible，simple and rapid，so it can provide technological support for real-time diagnosis and monitoring of WTD and DIV1D.