Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection.

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R