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 stonefruit 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 qualitycontrol testing of propagating material.

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

“…This property is particularly interesting for PDV and PNRSV as they are often present in mixed infections and their absence has to be controlled in phytosanitary certification programmes. A duplex protocol, based on primer pairs published by Kummert et al (2000), was previously optimized for the simultaneous detection of both viruses. The polyvalence of the primers was evaluated at the FUSAGx laboratory on 63 isolates of PDV and 101 isolates of PNRSV, respectively (Kummert et al 2000;Massart et al, unpublished data).…”

Development of RT-PCR tests for the routine detection of latent and ILAR viruses in fruit trees

“…PCR assay was simplified using crude plant extracts (MARINHO et al 1998;KUMMERT et al 1998KUMMERT et al , 2000KUMMERT et al , 2001. Samples (bark or leaves) were ground in an extraction buffer, diluted and added directly into the PCR mix.…”

Interlaboratory evaluation of two Reverse‐transcriptase Polymeric Chain Reaction‐based methods for detection of four fruit tree viruses

“…In this study, two duplex reverse-transcriptase (RT)-PCR methods, optimised (Massart et al, 2008) with previously published primers (Kummert et al, 2000), were evaluated. These methods were designed to detect four different fruit tree viruses belonging to the genus Ilarvirus (Apple mosaic virus, ApMV), Capillovirus (Apple stem grooving virus, ASGV), Trichovirus (Apple chlorotic leafspot virus, ACLSV) and Foveavirus (Apple stem pitting virus, ASPV).…”