Development of duplex PCR targeting RpmTEs and phcA genes for rapid and simultaneous detection of Ralstonia pseudosolanacearum in mulberry and other crops

Bacterial wilt disease is an important bacterial soil-borne disease that poses a considerable threat to the sericulture and other crop industries. In China, mulberry bacterial wilt disease is caused by Ralstonia pseudosolanacearum Mori (RPM), while in other crops, this disease is caused by R. pseudosolanacearum. Establishing a rapid detection method for bacterial wilt disease in mulberry and other crops is highly relevant for controlling its spread and damages. In the present study, we targeted the RPM-specific transposase RpmTEs gene (GenBank accession number: SAMN37721522) and R. pseudosolanacearum virulence genes transcriptional regulator phcA gene (GenBank accession number: AL646052.1) for developing a specific duplex PCR (dPCR) assay. The dPCR assay primers were designed, and 198 strains of R. pseudosolanacearum from nine different hosts were used to optimize the reaction system and amplification program. In addition, the RPM-dPCR detection assay was established to test its specificity, sensitivity, and practicality. In terms of specificity, the results showed that the RPM-specific detection primers RpmTEs-1F/R had a 2.27% higher accuracy rate compared to the MG67-F/R primers reported in previous literature. While the primers phcA-1F/R had a 5% higher accuracy rate than the AU759f/AU760r primers reported in previous literature. RPM-dPCR showed a DNA detection sensitivity of 1 pg/µL (equivalent to 1 × 104 CFU/mL) at RpmTEs-1F/R:phcA-1F/R = 0.25:0.5, an annealing temperature of 63 ℃ and 30 cycles. It was found to be highly specific, as it wasn’t affected by non-target bacteria. The RPM detection rate for mulberry bacterial wilt samples (125) was 64.8%, while R. pseudosolanacearum detection rate for other crops’ samples (143) was 46.9%. Compared to traditional isolation method, RPM and R. pseudosolanacearum detection rates were 56.8% and 44.1%, respectively. Traditional isolation methods had a 2.8–8.0% lower detection rate than RPM-dPCR assay. The established RPM-dPCR assay is simple, specific, sensitive, and efficient, as it can be simultaneously used for the rapid detection of bacterial wilt in mulberry and other crops.