ARS | Publication request: Development and Application of a Single-tube Immunocapture Real-Time PCR Technology for Sensitive Detection of a Panel of Viruses in Crop Plants

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2010
Publication Date: June 1, 2010
Citation: Ling, K., Feng, C., Xia, J.Q. 2010. Development and Application of a Single-tube Immunocapture Real-Time PCR Technology for Sensitive Detection of a Panel of Viruses in Crop Plants. Phytopathology. 100:S72.

Technical Abstract: Enzyme-linked immunosorbent assay (ELISA) is the most widely used technology for plant virus detection. Its sensitivity however may not be satisfactory in detecting viruses in tissues with early infection, seeds or woody plants. Recently, real-time PCR has been introduced for plant virus detection with higher sensitivity. Immunocapture (IC) real-time PCR is a combination of these two technologies which traps virus particles by immunocapture and washes away PCR inhibitors, followed by real-time PCR in the same tube for virus detection. The objectives of this study are to evaluate the factors affecting the immunocapture capabilities in microtubes and to develop sensitive real-time PCR to a panel of viruses. Sixteen microtube types were evaluated for their ability to immunocapture. Computer-assisted sequence analysis was used to select the most conserved genomic regions for primer and probe design. The target viruses captured on the microtubes were comparatively analyzed with ELISA, PCR and real-time PCR. Microtubes for the most effective immunocapturing of target viruses were identified. The IC real-time PCR could effectively detect 14 target viruses of tomato or pepper in crude tissue extract diluted up to 10<sup>-5</sup> or 10<sup>-7</sup>, which is approximately 1000 times higher than ELISA. Furthermore, multiplex IC real-time RT-PCR was developed for a simultaneous detection of 2-3 viruses. This technology has a potential to supplement ELISA for plant virus detection.