Sensitive detection of pea seed borne mosaic potyvirus by dot blot and tissue print hybridisation assays

Abstract

Dot blot and tissue print hybridisation assays were developed for the rapid and sensitive detection of pea seed borne mosaic virus (PSbMV). Radioactive (³²P-labelled) and non-radioactive (DIG-labelled) random primed cDNA probes representing the entire genome of PSbMV were used in hybridisation assays. A comparison of detection sensitivity was made between these probes and they were found equally sensitive. PSbMV was readily detected in dot blots by their homologous cDNA probes to 50 fg in purified preparations and to a 1 : 3125 dilution in purfied total nucleic acid or crude sap from infected plants. The sensitivity of heterologous probes was significantly less than that of homologous probes in purified preparation but nearly the same in purified total nucleic acid or crude sap from infected plants. The virus was also specifically detected in tissue squashes by tissue print hybridisation using cDNA probes. Non-radioactive probes were found suitable for PSbMV diagnosis without significant loss of detection sensitivity. There was no non-specific hybridisation against the healthy control in hybridisation assays using either radioactive or non-radioactive probes.