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

Abstract

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.