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Detection of apple chlorotic leaf spot virus using a 5′ nuclease assay with a fluorescent 3′ minor groove binder-DNA probe

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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.

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Acknowledgments

This work was supported by the General Directorate for Technologies, Research and Energy of the Wallonia Region, Belgium (DGTRE), in the framework of the research agreement N° 001/4542.

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