Direct real-time PCR detection of Plum pox virus in field surveys in Ontario

A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially...

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Bibliographic Details
Published in:Canadian journal of plant pathology 2008-04, Vol.30 (2), p.308-317
Main Authors: Kim, W.-S., Stobbs, L. W., Lehman, S. M., James, D., Svircev, A. M.
Format: Article
Language:English
Subjects:
Biological and medical sciences
diagnostic des maladies
disease diagnosis
détection des virus
Fundamental and applied biological sciences. Psychology
Phytopathology. Animal pests. Plant and forest protection
Plant viruses and viroids
Plum pox virus
polymerase chain reaction
Prunus
Prunus persica
réaction en chaîne de la polymérase
Sharka
Sharka disease
virus detection
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Summary:A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially developed for this purpose and named "direct pathogen extract buffer." Specific TaqMan primers and probes were designed for PPV detection: two sets specific to PPV D and M strains, respectively, and one for universal detection of PPV strains D, M, C, EA, and W. These primer and probe sets can be used singly or for duplex differentiation of D and M strains. Using Prunus spp. tissue infected with 30 known fruit tree viruses, the universal primer and probe set correctly identified PPV-infected and PPV-free samples, with no nonspecific cross-reactions. Based on endpoint analysis of the drtPCR reaction, a threshold cyle value of 36 was suggested as the maximum threshold to declare a sample positive for PPV. A comparative analysis comparing drtPCR and ELISA using 12 200 field samples revealed that drtPCR was approximately 100- to 1000-fold more sensitive than ELISA and was able to detect PPV at an earlier stage of infection than ELISA. The drtPCR is a valuable tool for PPV diagnosis and may also be applicable to other studies, including pathogen population dynamics and vector transmission efficiency.
ISSN:0706-0661
1715-2992
DOI:10.1080/07060661.2008.10540546