Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain

Abstract

The detection of soil-borne pathogens by quantitative PCR (qPCR) has been challenging due to the pronounced influence of soil type on DNA extraction and PCR reactions. In the present study, we developed a novel qPCR system and an internal sample process control (ISPC) strain, RsPC, for the detection of Ralstonia solanacearum species complex (RSSC), the pathogens causing bacterial wilt. Specific primers and TaqMan probe were designed based on analyses of 16S rRNA gene sequences from 581 Ralstonia genomes, and the RsPC was constructed by insertion of an artificial fragment, which consisted of two fragments from the kanamycin-resistant gene and the gfp gene, into the chromosome of a phylogenetically closely related strain, Ralstonia pickettii JCM 5969. The qPCR target sequences of RSSC and RsPC shared primers; however, different TaqMan probes were used to distinguish them from each other. The interaction assay between ISPC and target DNA showed no influence on sensitivity when their difference in concentration was <10 4 -fold. We tested 10 different soils with co-spiked RsPC and R. pseudosolanacearum LMG 9673 and found comparable recovery efficiencies (REs) of two strains in most samples, and the REs of LMG 9673 after correction by RsPC were closer to theoretical values. The RE of LMG 9673 improved most (2.98-fold) in a heavy clay soil. Similar results were obtained in three representative soils co-spiked with RsPC and LMG 9673 (at three concentrations), R. solanacearum NCPPB 325, and R. syzygii LLRS-1, respectively . The qPCR system and ISPC strain developed in this study could be applied for the accurate detection of RSSC in soil, and similar ISPCs can be developed in the future for other soil-borne animal and plant pathogens. IMPORTANCE DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.