Rapid And Homogenous Detection Of Apple Stem Pitting Virus By Rt-Pcr And A Fluorogenic 3 ' Minor Groove Binder-Dna Probe

Real-time PCR with fluorogenic hydrolysis probes (5' nuclease assay) is increasingly used for the detection of pathogens for diagnostic purposes. Nevertheless, the size of the probes, usually 25–40 nucleotides, might limit their use to detect pathogens with high genome variability between isolates, where an identical sequence cannot be found without multiple mismatches. In this report, we describe a 5' nuclease assay, to detect Apple stem pitting virus, based on the use of a shorter probe which is chemically modified with a minor groove binder in order to increase duplex stability and raise the melting temperature to a value suitable for real-time analysis. The short size of the probe, which is critical to target a conserved cluster sequence of 14 nucleotides in the RNA polymerase gene, circumvents the genome variability of the virus. The assay correlates at 96 percent with gel analysis and is more reliable than biological indexing to detect Apple stem pitting virus field isolates. It is fast and fully compatible with automation, and therefore particularly suitable for plant certification.