Ypt1 gene-based recombinase polymerase amplification assay for Phytophthora capsici and P. tropicalis detection in black pepper

Foot rot caused by Phytophthora is one of the major diseases of black pepper (Piper nigrum L.). Accurate and timely diagnosis of the disease is crucial for its successful management. Although PCR and qPCR assays are used for detection, the cost and time required to perform these assays are high. Recombinase polymerase amplification (RPA) assay has the advantage of minimal assay time and it is performed under isothermal conditions. Hence, RPA assay was developed for the detection of P. capsici and P. tropicalis and compared with newly developed end-point PCR test. Out of three sets of primers analyzed, a primer set based on the Ypt1 gene successfully amplified a 230/231 bp product. Optimum amplification of RPA products were observed when the assay was performed at 37 °C with 14 mM magnesium acetate for 40 min. Sensitivity analysis using serial dilutions indicated that RPA is 10 times more sensitive than end-point PCR. During specificity analysis, non-specific bands were observed with other Phytophthora species, and hence the assay was further refined with betaine wherein addition of 1.0 M betaine avoided amplification of non-specific bands. The optimized RPA assay could detect Phytophthora from infected black pepper leaf, stem and root using both purified DNA and crude extracts. The end-point PCR test successfully differentiated the two species of Phytophthora in a validation test. These results indicate the robustness of the developed end-point PCR and RPA assays and its potential application in detection and differentiation of P. capsici and P. tropicalis infecting black pepper.