Articles
DEVELOPMENT OF ROUTINE RT-PCR ELOSA TESTS FOR FRUIT TREE CERTIFICATION
Article number
550_3
Pages
45 – 52
Language
English
Abstract
Current developments in certification procedures for fruit tree multiplication material require the availability for rapid, sensitive, reliable and user’s friendly detection protocols applicable for routine testing.
Our research concerns the possible use of RT-PCR for the detection of the viruses listed for the virus-tested material of pome- and stone-fruit multiplication material in Belgium.
Although RT-PCR allows to reach the demand for rapidity and sensitivity, the usual protocols relying on the use of purified nucleic acid preparations as template, and agarose gel electrophoresis for detection, are not appropriate for routine use.
The use of carefully optimised RT-PCR reactions allowing this reaction to be performed on crude extracts of fruit tree tissues associated to a colorimetric detection of amplification products in microtiter plates would render this technique easier to perform, and thus more adapted for routine testing.
Considering the high specificity needed for certification, a protocol using hybridisation of specific amplification products between labelled capture and detection probes has been retained.
This has been achieved with the RT-PCR-ELOSA diagnosis system of Lambdatech S.A.(Namur, Belgium) using sandwich hybridisation of amplification products to specific detection probe, labelled with biotin, and capture probe phosphorylated at its 5’ end, covalently linked to microtiter plates.
Analysis of available sequence data has been used to define primers and probes responding to the constraints of size and structure of the Lambdatech system for the RT-PCR-ELOSA detection of Apple chlorotic leafspot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV). The RT-PCR-ELOSA detection tests defined for these viruses are under evaluation.
Our research concerns the possible use of RT-PCR for the detection of the viruses listed for the virus-tested material of pome- and stone-fruit multiplication material in Belgium.
Although RT-PCR allows to reach the demand for rapidity and sensitivity, the usual protocols relying on the use of purified nucleic acid preparations as template, and agarose gel electrophoresis for detection, are not appropriate for routine use.
The use of carefully optimised RT-PCR reactions allowing this reaction to be performed on crude extracts of fruit tree tissues associated to a colorimetric detection of amplification products in microtiter plates would render this technique easier to perform, and thus more adapted for routine testing.
Considering the high specificity needed for certification, a protocol using hybridisation of specific amplification products between labelled capture and detection probes has been retained.
This has been achieved with the RT-PCR-ELOSA diagnosis system of Lambdatech S.A.(Namur, Belgium) using sandwich hybridisation of amplification products to specific detection probe, labelled with biotin, and capture probe phosphorylated at its 5’ end, covalently linked to microtiter plates.
Analysis of available sequence data has been used to define primers and probes responding to the constraints of size and structure of the Lambdatech system for the RT-PCR-ELOSA detection of Apple chlorotic leafspot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV). The RT-PCR-ELOSA detection tests defined for these viruses are under evaluation.
Publication
Authors
J. Kummert, M. Vendrame, P. Lepoivre, S. Steyer
Keywords
ACLSV, ASGV, ASPV, PDV, PNRSV, RT-PCR, Colorimetric detection, Crude extracts
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