ARS | Publication request: Evaluation of LNA, MGB and non-modified DNA probes to improve the detection limit of TaqMan real-time PCR assay for Pantoea stewartii subsp. stewartii

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 4, 2007
Publication Date: August 1, 2007
Citation: Fessehaie, A., Block, C.C., Shepherd, L.M., Misra, M.K. 2007. Evaluation of LNA, MGB and non-modified DNA probes to improve the detection limit of TaqMan real-time PCR assay for Pantoea stewartii subsp. stewartii [abstract]. American Phytopathological Society Annual Meeting. 97:S35.

Technical Abstract: The goal of this study was to compare the sensitivity and amplification efficiency of the TaqMan assay using locked nucleic acid (LNA), minor groove binder (MGB) ligands and non-modified DNA probes. In monoplex or single target TaqMan assays for P. stewartii subsp. stewartii, LNA and MGB probes improved detection sensitivity by 10 to 100 fold compared to the conventional probe. When an internal positive control was added to the real-time PCR assay, the now duplex TaqMan PCR assay containing the MGB probe performed slightly better in terms of detection efficiency than the LNA probe. Both were superior to the non-modified or conventional probe. Thus, the novel TaqMan PCR assay with the MGB probe improved detection sensitivity and efficiency compared to the other TaqMan PCR assays for P. s. subsp. stewartii. Additionally, the TaqMan real-time PCR assay with MGB probe was successfully employed in the development of a multiplex PCR assay for simultaneous detection of P. s. subsp. stewartii and Stenocarpella maydis in a single PCR reaction tube.