A Comparison of Standard and High-Fidelity PCR: Evaluating Quantification and Detection of Pathogen DNA in the Presence of Orchid Host Tissue.

Abstract

The polymerase chain reaction (PCR) has been used with increasing frequency for detecting and identifying plant pathogens. Although PCR is sensitive, research has shown that amplification of target microbial DNA from within another organism, such as an arthropod or plant, can be inhibited by the presence of host DNA. In this study, the sensitivity of standard and high-fidelity PCR, which incorporates a second DNA polymerase with proofreading ability, to detect and amplify DNA from the fungal pathogen Pseudocercospora odontoglossi while in the presence of Cattleya orchid DNA, was compared. Different dilutions of plasmids containing internal transcribed spacer (ITS)1, 5.8S, and ITS2 rDNA from P. odontoglossi were spiked with Cattleya orchid plant DNA. The high-fidelity PCR could detect and amplify as few as 207 plasmids containing the fungal DNA, whereas the standard PCR required over 200 million copies. The high-fidelity PCR was more efficient than conventional PCR in detecting Sclerotium rolfsii and a Dickeya sp. from freshly inoculated orchid plants, demonstrating its increased sensitivity in early detection of fungal and bacterial pathogens that are difficult to discriminate early in disease development.