Imported cowpea seeds were detected with growing test,ELISA assay and RT-PCR method.The ELISA results showed that cowpea seedlings with symptoms reacted positively with antibody against Southern bean mosaic virus(SBMV).The 979 bp of fragment could be amplified from two positive ELISA samples using primers specific for Southern cowpea mosaic virus(SCPMV),and the sequence determination results proved that the pathogen existing in imported cowpea seeds was SCPMV.The positive ELISA results with SBMV antibody could be further confirmed by RT-PCR amplification with specific primers designed to amplify the coat protein gene and 3’ noncoding region of SCPMV and SBMV.The RT-PCR method presented here was suitable for molecular identification of SBMV and SCPMV in entry-exit plant quarantine laboratories.
The nucleotide sequence of small coat protein(CPS) gene of Broad bean stain virus(BBSV) was determined and compared with other comoviruses.The CPS gene of BBSV consisted of 687 nucleotides and encodes a putative protein of 228 amino acid residues.The CPS sequence of BBSV and those of other comoviruses shared identities of 36.5%-58.9% and 35.2%-70.3% at the nucleotide and amino acid levels,respectively.The RT-PCR method specific for BBSV detection was developed based on the determined CPS sequence.The RT-PCR assay presented here allows,for the first time,rapid and specific detection of BBSV.
