Detection of three pear viruses by multiplex RT-PCR assays with co-amplification of an internal control

Apple stem pitting virus, Apple chlorotic leaf spot virus and Apple stem grooving virus are economically important and common pathogens of pear and apple cultivars. All are symptomless in commercial pear and apple cultivars. Two multiplex RT-PCR methods with co-amplification of an internal control from a plant mRNA sequence were used to detect these three viruses simultaneously. The approaches, including what materials were to be selected and how to extract nucleic acids, were designed to improve detection efficiency. Detection assays, using bark chips of 1- or 2-year-old pear branches, could be performed both in the growing season and in dormant trees. A specific internal RNA control, which could be amplified from total nucleic acids, was co-amplified with the target viruses. Therefore, purified RNA as an amplification template was not necessary. Isolation of nucleic acids from bark chips was more successful than isolation from leaves. Three virus-specific primer pairs were designed to allow two multiplex RT-PCR amplifications accompanied by one pair of primers of the mitochondrial nad5 gene. Four amplification products including three viruses and an internal control were sequenced to prove the specificity of all primer pairs. By using a nad5 gene in mitochondrial mRNA of the apple as an internal control of co-amplification, these methods reduced the risk of false negative results that may occur with routine RT-PCR assays.