Development and application of triplex PCR detection system for pepper soil-borne pathogens

【Objective】 In this study, a multiplex PCR method was developed for early and rapid detection of Phytophthora capsici, Sclerotium rolfsii and Ralstonia solanacearum at the same time, and provide technical support for early diagnosis of soil-borne disease pathogens in pepper field.【Method】 Three soil-borne disease pathogens of pepper, P. capsici, S. rolfsii and R. solanacearum, were selected, and three pairs of specific primers, PCA1F/PCA1R, SCR-F/SCR-R and RalfliC-F/RalfliC-R, were screened with reference to relevant papers. The optimal reaction system was explored by setting different primer concentrations, annealing temperatures, cycle numbers and extension times to establish a triplex PCR detection method, and that was validated based on the actual diseased samples in the field.【Result】 In the optimized triplex PCR system 25.0 μL, the concentrations of the primer pairs of PCA1F/PCA1R, SCR-F/SCR-R and RalfliC-F/RalfliC-R were 0.24, 0.24 and 0.28 μmol/L, respectively;DNAtemplates were 10 ng each;2×Multiplex PCR Mix 12.5 μL, added ddH₂O to make up to 25.0 μL. The optimal amplification procedure was as follows:pre-denaturation at 94℃ for 1 min followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 55.8℃ for 30 s and extension at 72℃ for 45 s; finally extension at 72℃ for 10 min. Established triplex PCR system could amplify specific fragments of P. capsici, S. rolfsii and R. solanacearum with the length of 352, 540 and 724 bp, respectively, and with the sensitivity of 10^-1 ng/μL. Using the above reaction system, 54 pepper and soil samples collected from different counties and cities in Jiangxi were detected, and the detection rate of the samples was 100%.【Conclusion】 The established triplex PCR is able to rapidly and accurately detect P. capsici, S. rolfsii and R. solanacearum in diseased plants and soil in the field. It can be used for early prevention and epidemic monitoring of various soil borne diseases of pepper.