A new virus detection method was developed by combining hybridization capture and real-time PCR technology with the sample of Tomato ringspot nepovirus.High purified DNA or RNA was crucial for the real-time PCR detection as the inhibitors of PCR reaction in template and might cause the false-negative cases.A probe was covalent bound to the surface of PCR tube for the DNA capture followed by real-time PCR detection.The steps of nucleic acids preparation and detection were both done in one the same tube.This method reduced the possibility of contamination as well increased the detection efficiency.It can be applied in plant virus detection especially for the sample with many inhibitors of PCR reaction.
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.
