Detection Of Apple Chlorotic Leaf Spot Virus Using A 5 ' Nuclease Assay With A Fluorescent 3 ' Minor Groove Binder-Dna Probe

Abstract 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.