Improved detection of prunus necrotic ringspot virus by the polymerase chain reaction

The reverse transcription - polymerase chain reaction (RT-PCR) technique was used for detection of prunus necrotic ringspot virus in dormant peach trees which tested negative by ELISA. Total RNA extracted from bark tissue, using a lithium chloride based method, were used for reverse transcription and subsequent amplification of viral sequences. The PCR product, about 300 base pairs in size, was analyzed by gel electrophoresis and visualized by ethidium bromide staining. In some cases, PCR products were not clearly visible in the stained gel and became distinct only following hybridisation with a P-32-labelled virus specific probe. The RT-PCR assay described in this paper is sensitive enough for detection of PNRSV in dormant woody bark tissue and could be incorporated into testing protocols during post-entry quarantines for rapid initial screening of imported budwood and in virus elimination programs.