Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR.
Plum pox virus (PPV), a destructive and economically devastating pathogen of Prunus species, was recently discovered in Pennsylvania and Canada. Current containment efforts involve eradication of infected trees based on ELISA surveys, which are laborious and less sensitive than PCR-based techniques. A real-time, fluorescent, reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of PPV in the Smart Cycler (Cepheid). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10-20 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by visualization with ethidium-bromide. PPV was detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantitation of viral template. The real-time PCR assay is a valuable tool for PPV detection and liter quantification in field or laboratory settings.
Schneider
William L
WL
USDA-ARS Foreign Disease Weed Science Research Unit, Fort Detrick, MD 21702, USA. wschneider@fdwsr.ars.usda.gov
Sherman
Diana J
DJ
Stone
Andrew L
AL
Damsteegt
Vernon D
VD
Frederick
Reid D
RD
D001483:Base Sequence; D005453:Fluorescence; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D018517:Plant Roots / Q000821:virology; D018547:Plant Stems / Q000821:virology; D017801:Plum Pox Virus / Q000235:genetics* / Q000302:isolation & purification*; D027861:Prunus / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012367:RNA, Viral