Detection of apple chlorotic leaf spot virus using a 5' nuclease assay with a fluorescent 3' minor groove binder-DNA probe.
The development of a real-time 5' nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3' minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe/target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.
Salmon
Michel A
MA
Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés 2, 5030 Gembloux, Belgium.
Vendrame
Marina
M
Kummert
Jean
J
Lepoivre
Philippe
P
D001483:Base Sequence; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D005456:Fluorescent Dyes; D027845:Malus / Q000821:virology*; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes