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.

由疫霉菌引起的足腐病是黑胡椒（Piper nigrum L.）的主要疾病之一。准确，及时地诊断出该疾病对于成功治疗至关重要。尽管使用PCR和qPCR测定法进行检测，但是执行这些测定法所需的成本和时间很高。重组酶聚合酶扩增（RPA）分析的优点是分析时间最短，并且可以在恒温条件下进行。因此，开发了RPA测定法来检测辣椒辣椒和热带假单胞菌，并将其与新开发的终点PCR测试进行比较。在分析的三组引物中，基于Ypt1的引物组该基因成功扩增出230/231 bp的产物。当在37°C下用14 mM乙酸镁进行40分钟分析时，可以观察到RPA产物的最佳扩增。使用系列稀释液的灵敏度分析表明，RPA的灵敏度是终点PCR的10倍。在特异性分析过程中，与其他疫霉菌一起观察到非特异性条带，因此用甜菜碱进一步完善了测定方法，其中添加1.0 M甜菜碱可避免非特异性条带的扩增。优化的RPA测定法可以使用纯化的DNA和粗提物从受感染的黑胡椒叶，茎和根中检测疫霉菌。终点PCR测试成功地区分了疫霉属的两种在验证测试中。这些结果表明，已开发的终点PCR和RPA分析的稳健性及其在检测和鉴别感染黑胡椒的辣椒和热带假单胞菌中的潜在应用。