Location: National Germplasm Resources LaboratoryTitle: Development a of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic virus) Author
Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2013
Publication Date: N/A
Citation: Interpretive Summary: Viruses and viroids are important pathogens of stone fruit (peach, plum, almond, etc.) trees and can be easily, but unknowingly, spread as vegetative cuttings are distributed worldwide. Most countries have quarantines that regulate the importation of stone fruit clonal plant material. Diagnostic testing is required to infer the absence of quarantine pathogens before the material can enter the country. Reliable and sensitive detection methods are needed to support these activities. We report a technique to detect simultaneously three pathogens that infect stone fruit trees: Asian prunus viruses, Plum bark necrosis stem pitting associated virus, and Peach latent mosaic viroid. The first two are recently discovered viruses and the third appears to be the most common viroid infecting stone fruit trees. The assay uses RT-PCR primers to amplify target regions of the pathogen genomes and a fluorescent probe to detect the amplification products. The technique is highly automated and is performed in a single tube, factors that reduce the opportunities for contamination. Testing for three pathogens in one reaction also reduces costs. The procedure is sensitive and reliable and can be used by programs that test stone fruit plant material for these three pathogens.
Technical Abstract: Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR assay was developed for the simultaneous detection and identification of these three pathogens. The protocol includes amplification and detection of a fluorogenic cytochrome oxidase gene (COX) as an internal control. The results of the multiplex TaqMan RT-PCR assay correlated with those from conventional RT-PCR, with a 10-fold increase in sensitivity in the multiplex real-time format. The efficiency and accuracy of the assay was evaluated by testing stone fruit trees from positive control collections and several orchard locations. Several mixed infections of target pathogens were detected in peach orchard samples. This assay is simple, rapid and cost-effective and can be used by quarantine and certification programs where numerous stone fruit trees need to be tested for all three pathogens.