A new combination of RT-PCR and reverse dot blot hybridization for rapid detection and identification of potyviruses

Three degenerate primers, located at the NIb and CP gene regions, were designed for potyvirus detection. Using these primer pairs, 1.0-1.2 kb cDNA fragments of the 3′-terminal region of six potyviruses were successfully amplified from infected plant tissues. RT-PCR products were sequenced and found to be derived from the expected viruses. To identify further these potyviruses, sequences located between the 3′ end of the NIb gene and the 5′ end of the CP gene were chosen to design a series of species-specific probes. The probes were prepared by PCR with species-specific primers, immobilized onto nylon membrane, and then hybridized with DIG-labeled RT-PCR products amplified by potyvirus degenerate primers. The results suggested that species-specific cDNA probes plus reverse dot blot hybridization was able to identify correctly different species of potyviruses in single as well as mixed infections.