Detection of cowpea aphid-borne mosaic virus (CABMV) in cowpea by reverse transcription polymerase chain reaction

Abstract

The occurrence of CABMV on cowpea (Vigna unguiculata L. Walp) in Uganda was described recently in several studies. This study developed and optimised a reverse transcription polymerase chain reaction (RT-PCR) based assay for the detection of CABMV in leaf samples, and compared it to previous RT-PCR and ELISA assays. Use of the forward primer (CABFF1, 5'- GGT AAC AAY AGT GGR CAA CC-3') and the reverse primer (CABRR1, 5'- CTG AGC ACT CMA ACC GGG-3') yielded a product of ~ 1,642 bp. Amplicon sequencing and subsequent BLASTN analysis showed that Ugandan isolates were 89.3-94.3% identical indicating they belong to the same strain of CABMV. Phylogenetic analysis also placed the Ugandan isolates in the same cluster different from other isolates but closer to those from Burkina Faso. However, the previously reported RT-PCR assay (GF/GR primer pair) did not give the expected PCR fragment (221 bp) and gave no virus hits upon amplicon sequencing and sequence analysis. The ELISA assay did not differentiate between positive and negative samples. The newly developed RT-PCR assay for detecting CABMV, described in this study, has important applications for plant quarantine, resistance breeding, host range studies as well as epidemiological studies for the control of CABMV in the country.

Keywords: NCM-ELISA, RT-PCR, sequencing, Vigna unguiculata