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Title: Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus

Author
item Ling, Kai-Shu
item Wechter, William - Pat
item Jordan, Ramon

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2007
Publication Date: 7/26/2007
Citation: Ling, K., Wechter, W.P., Jordan, R.L. 2007. Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus. Journal of Virological Methods 144:65-72.

Interpretive Summary: Pepino mosaic is an emerging disease on greenhouse tomato. The disease is now widespread on greenhouse tomatoes in Europe, North America, South America, and Asia. In the U.S., this disease is threatening the profitability of greenhouse tomato production in many states, including Arizona, California, Colorado, and Texas. The seed borne nature of Pepino mosaic virus (PepMV) in commercial tomato seed and the increasing world trade of hybrid tomato seeds may have contributed to this worldwide outbreak. Although serological and biological methods for the detection of PepMV are available, they may not offer the sensitivity for a reliable detection of low level of PepMV in tomato seed. In the present study, we developed a sensitive molecular detection technique, Real-time reverse transcription-polymerase chain reaction (RT-PCR), which would allow for an efficient detection of genetically diverse PepMV strains. The Real-time RT-PCR was able to detect PepMV in one infested seed in 1,000. This level of sensitivity showed that the Real-time RT-PCR developed in the present study is useful for routine seed health assays.

Technical Abstract: A Real-time reverse transcription-polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse PepMV isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single conserved TaqMan probe to broaden its reaction to cover all PepMV strains. This duo-primer Real-time RT-PCR assay was evaluated against US1, US2, Ch1, Ch2 and 26 field isolates collected from six major commercial tomato greenhouse facilities in U.S. and Canada in 2006. Under optimum reaction conditions, sensitivity of the detection was as low as 100 fg of purified viral RNA. This assay was also evaluated for its efficiency in detecting PepMV in various levels of contaminated seed samples. Using immunocapture sample preparation, The Real-time RT-PCR was able to detect PepMV in one infested seed in 1,000. This level of sensitivity indicated that the one-step immunocapture duo-primer TaqMan Real-time RT-PCR developed in the present study could be used for routine seed health assays.