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United States Department of Agriculture

Agricultural Research Service

Title: Quantification of Positive and Negative Strand Ratios for Plum Pox Virus by Real-Time Fluorescent Reverse Transcription-Pcr

Authors
item Schneider, William
item Stone, Andrew
item Sherman, Diana
item Damsteegt, Vernon

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 10, 2005
Publication Date: N/A

Interpretive Summary: Plum pox potyvirus (PPV), a destructive and economically devastating disease of peaches and plums, was recently discovered in Pennsylvania. The efforts to eradicate this disease rely on efficient and sensitive detection of the virus. Current detection surveys use antibody-based methods, which are laborious and less sensitive than molecular techniques. A real-time molecular assay was developed for the detection of. The methods developed are reproducible, specific to PPV, and sensitive enough to detect trace amounts of the virus. The assay is faster and more sensitive than either traditional assays currently in use for surveys. PPV was effectively detected from leaves, stems, buds flowers and roots of multiple hosts. In addition, the assay can be used to quantify the amount of virus in a given sample, which is important to future research.

Technical Abstract: Plum pox potyvirus (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 quantification of viral template. The real-time PCR assay is a valuable tool for PPV detection and titer quantification in field or laboratory settings.

Last Modified: 7/25/2014
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