Development of routine RT-PCR tests for certification of fruit tree multiplication material*

Current developments in certification procedures for propagating material require the availability of rapid, sensitive, reliable and user-friendly detection protocols applicable for routine testing. Our research concerns the possible use of reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of the pathogens listed for virus-tested pome and stone-fruit propagating material in Belgium. Although RT-PCR satisfies the need for rapidity and sensitivity, the usual protocols relying on the use of purified nucleic acid preparations as template and ethidium bromide-stained agarose gels for detection are not appropriate for routine use. We therefore first optimized the parameters and cycling conditions of the RT-PCR reactions to allow direct use of crude extracts of either leaf or bark material as a template. Sandwich hybridization between a covalently linked capture probe and a biotinylated detection probe was then used for the detection of the specific amplicons (Lambdatech S.A. kits in development). These assays have the sensitivity and specificity of the RT-PCR, enhanced by sandwich hybridization with specific probes, and ease of sample preparation and detection of the amplicons. They make it possible to analyse a great number of samples and are thus well adapted for routine quality-control testing of propagating material.