Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides , causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.