TY - JOUR
T1 - Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR
AU - Schneider, William L.
AU - Sherman, Diana J.
AU - Stone, Andrew L.
AU - Damsteegt, Vernon D.
AU - Frederick, Reid D.
PY - 2004/9/1
Y1 - 2004/9/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=3242796517&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3242796517&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2004.04.010
DO - 10.1016/j.jviromet.2004.04.010
M3 - Article
C2 - 15234814
AN - SCOPUS:3242796517
SN - 0166-0934
VL - 120
SP - 97
EP - 105
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -