Brief communication 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. © 2002 Elsevier Science B.V. All rights reserved.