Development of a TaqMan real-time RT-PCR assay for detection of covert mortality nodavirus (CMNV) in penaeid shrimp

Abstract Covert mortality nodavirus (CMNV) is an RNA virus that infects shrimp and is responsible for economic losses in the shrimp aquaculture industry in several countries of Southeast Asia and China. Detection of the disease at an early stage can enhance proper farm management. However, the recent disease monitoring technique of confining nested reverse-transcription PCR (RT-PCR) is time consuming and does not determine the viral load in infected shrimp. This study develops a more efficient technique for the detection of CMNV in infected shrimp using TaqMan probe-based real-time RT-PCR. The procedure comprises the isolation of CMNV from infected shrimp and the isolation of the messenger RNA to construct a complementary DNA template for PCR. Viral detection viability was tested using both nested RT-PCR and real-time RT-PCR. The results showed that real-time RT-PCR is highly sensitive in terms of minimal viral copy numbers compared to nested RT-PCR results. Real-time RT-PCR has viral detection capability as small as 1 copy number of the CMNV plasmid while conventional RT-PCR and nested RT-PCR recognize minimal detectable numbers of 10,000 and 100 viral copies in shrimp sample, respectively. The results from real-time RT-PCR showed the viral load in shrimp samples varied from 4.27 to 6.53 × 10 6 copies number, while nested RT-PCR recognize minimal detectable numbers of 281 viral copies. The real-time RT-PCR technique developed in this study is advantageous because it is less time consuming compared to the nested RT-PCR technique, works well in viral load analysis and can exclude cross-reaction with other shrimp RNA viruses when tested against TSV, YHV, and IMNV. Statement of relevance This study developed methods for CMNV detection in penaeid shrimp.