Detection of Viruses in Apples and Pears by Real Time RT-PCR Using 5'-Hydrolysis Probes

Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV) are common in apples and pears and the main targets of virus elimination from propagation material. The objective of this work was to design primers and probes for a real time RT-PCR protocol for detection of the four above viruses. FAM/TAMRA-labeled probes and primers were designed by searching for highly conserved nucleotide regions in the coat protein gene of the four viruses. Infection levels in analyzed apple samples were 92.6, 96.4, 100 and 88% for ASGV, ASPV, ACLSV and ApMV, respectively. In pears, all pre-existing ASPV infections were detected. Viral infections were confirmed in a selection of commercial cultivars of apples and pear scions, and quince rootstocks, demonstrating the sensitivity and reliability of the designed primers and probes. Real time RT-PCR using 5'-labeled probes is suitable for checking sanitary quality as a routine test in certification programs