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United States Department of Agriculture

Agricultural Research Service

Title: Realtime Ic Rt-Pcr Detection of Pepino Mosaic Virus on Tomato Seed and Plant Tissues in a Single Tube

item Ling, Kai-Shu

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 30, 2005
Publication Date: July 30, 2005
Citation: Ling, K. 2005. Realtime immunocapture RT-PCR detection of Pepino Mosaic virus on tomato seed and plant tissues in a single tube [abstract]. Phytopathology 95 (6):S61.

Technical Abstract: Pepino mosaic virus (PepMV) is an emergent disease on greenhouse tomato in Europe and North America. The seed-borne nature of PepMV, coupled with easy mechanical transmission, make tomato seed a potential inoculum source. The current seed health test for PepMV relies mainly on ELISA. For a seed lot with low level of viral contamination, ELISA may not be sensitive enough. RT-PCR is a good alternative. Immunocapture (IC) was used to capture virus particles with PepMV-specific antibody absorbed to PCR tubes. The seed or tissue extract was prepared with a general ELISA sample extraction buffer without RNA isolation. This immunocapture procedure enriched virus particles while eliminating potential PCR-inhibiting compounds. Utilizing Realtime, IC, RT-PCR allowed us to perform an entire assay, from sample extraction to amplicon detection, in a single reaction tube. The PepMV specific primer set and TaqMan probe were designed based on the consensus sequence generated from a multiple genome sequence alignment of the available PepMV sequences plus two additional isolates characterized recently in our laboratory. The single-tube format eliminated the potential for cross contamination during sample transfer in RT-PCR. Sensitivity of the detection could reach up to one in ten billion (w/v) dilution in tomato seed or tissue samples in comparison with only one in one hundred in an ELISA.

Last Modified: 4/22/2015
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