Development of a multiplex RT-PCR assay for simultaneous detection of viruses in cowpea

The diagnosis of viral diseases in cowpea is crucial for the efficient implementation of control measures. A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of cowpea severe mosaic virus (CPSMV), cucumber mosaic virus (CMV), and cowpea mild mottle virus (CPMMV) in cowpea. The targeted viruses produced the expected amplicons, namely 300 bp for CMV, 500 bp for CPMMV, and 890 bp for CPSMV. The relative analytical sensitivities of the assay was determined through probit analysis of serial dilutions (10⁻¹ to 10⁻⁶), with detection limits at 95% probability established as 10^− 2 for all viruses in multiplex RT-PCR. The performance of multiplex RT-PCR was confirmed by uniplex RT-PCR for each target virus. The multiplex RT-PCR of field cowpea leaf samples revealed a high incidence of CMV and CPSMV, and no infection by CPMMV. Approximately 24% of the samples were simultaneously infected with CPSMV and CMV. No other comovirus, besides CPSMV, was detected, when the samples were tested with degenerate primers for comoviruses by uniplex RT-PCR. This assay streamlines the diagnosis of CPSMV, CMV, and CPMMV by offering a cost-effective and efficient tool. Additionally, it supports epidemiological studies of these pathogens, providing critical data to inform effective control strategies.