Abstract：
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.
摘要：
细粒棘球蚴（Eg）和多房棘球蚴（Em）是两种最广泛流行的棘球蚴病。已经开发了几种诊断方法来检测Eg和Em。然而,一些限制,比如耗时，需要昂贵的仪器，或表现出低灵敏度，使这些方法不适合现场检测。在这项研究中,建立了双RPA测定法来检测和区分Eg和Em。底漆浓度比，反应时间,优化了双RPA的反应温度。结果表明,Eg:Em的引物浓度比为400nM:400nM,在38°C反应20分钟可获得最佳的扩增效率。敏感性,特异性，和重复性的测定也进行了测试。测定对Eg和Em的检测极限为10拷贝/μL。通过测试十种寄生核酸，该测定法显示出合理的特异性。试验的批内和批间变异系数低于10%，这表明该测定的可靠可重复性。最后,为了验证双RPA测定的性能，通过使用86个临床核酸样本与实时PCR进行比较。双RPA与TaqManreal-timePCR的Eg符合率为96.51%,双RPA与TaqManreal-timePCR的Em符合率为98.84%,表明其准确临床诊断的潜力。因此,本研究建立了一种快速、灵敏的双RPA检测方法，可以在一个反应管中快速检测和区分Eg和Em，为该领域的棘球蚴病检测提供了一种新的检测方法。
