Dual-RPA assay for rapid detection and differentiation of E.granulosus and E.multilocularis.

Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em) are the two most widely prevalent types of echinococcosis. Several diagnostic methods have been developed for detecting Eg and Em. However, some limitations, such as being time-consuming, needing expensive instruments, or exhibiting low sensitivity, make these methods unsuitable for on-site detection. In this study, a dual-RPA assay was established to detect and differentiate Eg and Em. The primer concentration ratio, reaction time, and reaction temperature of the dual-RPA were optimized. The result showed that the primer concentration ratio of Eg:Em was 400 nM:400 nM, and the best amplification efficiency was obtained by reacting at 38 °C for 20 min. The sensitivity, specificity, and repeatability of the assay were also tested. The assay's detection limit for both Eg and Em was 10 copies/μL. The assay showed reasonable specificity by testing ten parasitic nucleic acids. The assay's intra- and inter-batch coefficients of variation were below 10%, which indicates robust reproducibility of the assay. Finally, to validate the performance of the dual-RPA assay, it was compared with real-time PCR by using 86 clinical nucleic acid samples. The coincidence rate of Eg between dual-RPA and TaqMan real-time PCR was 96.51%, and the coincidence rate of Em between dual-RPA and TaqMan real-time PCR was 98.84%, indicating its potential for accurate clinical diagnosis. Therefore, this study established a rapid and sensitive dual-RPA assay that can rapidly detect and differentiate Eg and Em in one reaction tube and provided a new assay for the detection of echinococcosis in the field.