Articles
DEVELOPMENT OF COLORIMETRIC RT-PCR TESTS FOR APPLE STEM GROOVING VIRUS DETECTION IN APPLE TREES
Article number
472_9
Pages
105 – 112
Language
Abstract
RT-PCR technology is becoming available for diagnosis of pome fruit virus detection in their natural hosts, the amplified products being usually visualized after electrophoresis in ethidium bromide stained gels.
Although highly sensitive, this technology is not amenable to routine testing of large number of samples, i.e. for certification purposes.
The aim of our work was to develop a colorimetric RT-PCR (or RT-PCR ELISA) test that combines the PCR sensitivity and the convenience of ELISA. The RT-PCR amplified products were either directly labeled with digoxigenin (DIG) or, for better specificity, unlabeled amplification products were hybridized to an internal DIG-labeled detection probe.
In both systems, a biotin-labeled probe was used for trapping the amplification products on streptavidin-coated microplate strips.
Revelation was performed with an alkaline phosphatase labeled anti-DIG conjugate (DIG Detection Kit, Boehringer Mannheim). The results showed high sensitivity for the detection of ASGV in apple tissues.
The importance of specific primers allowing high cycling temperatures, in relation with the origin and purification status of the plant extracts used for RT-PCR, and the roles of capture and detection probes on the specificity of the colorimetric RT-PCR are discussed.
Although highly sensitive, this technology is not amenable to routine testing of large number of samples, i.e. for certification purposes.
The aim of our work was to develop a colorimetric RT-PCR (or RT-PCR ELISA) test that combines the PCR sensitivity and the convenience of ELISA. The RT-PCR amplified products were either directly labeled with digoxigenin (DIG) or, for better specificity, unlabeled amplification products were hybridized to an internal DIG-labeled detection probe.
In both systems, a biotin-labeled probe was used for trapping the amplification products on streptavidin-coated microplate strips.
Revelation was performed with an alkaline phosphatase labeled anti-DIG conjugate (DIG Detection Kit, Boehringer Mannheim). The results showed high sensitivity for the detection of ASGV in apple tissues.
The importance of specific primers allowing high cycling temperatures, in relation with the origin and purification status of the plant extracts used for RT-PCR, and the roles of capture and detection probes on the specificity of the colorimetric RT-PCR are discussed.
Authors
J. Daniels, V.L.A. Marinho, J. Kummert, P. Lepoivre
Keywords
Online Articles (101)