College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou, 256600, China.
Microb Pathog. 2024 Apr;189:106600. doi: 10.1016/j.micpath.2024.106600. Epub 2024 Feb 29.
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.
细粒棘球绦虫（Eg）和多房棘球绦虫（Em）是两种最广泛流行的包虫病类型。已经开发了几种诊断方法来检测 Eg 和 Em。然而，一些限制，如耗时、需要昂贵的仪器或灵敏度低，使得这些方法不适合现场检测。在本研究中，建立了一种双重 RPA 检测方法来检测和区分 Eg 和 Em。优化了双重 RPA 的引物浓度比、反应时间和反应温度。结果表明，Eg:Em 的引物浓度比为 400 nM:400 nM，在 38°C 反应 20 min 可获得最佳扩增效率。还测试了该检测方法的灵敏度、特异性和重复性。该检测方法对 Eg 和 Em 的检测限均为 10 拷贝/μL。该检测方法通过测试十种寄生虫核酸显示出合理的特异性。该检测方法的批内和批间变异系数均低于 10%，表明该检测方法具有良好的重现性。最后，通过使用 86 个临床核酸样本，将双重 RPA 检测与实时 PCR 进行比较，验证了双重 RPA 检测方法的性能。双重 RPA 与 TaqMan 实时 PCR 检测 Eg 的符合率为 96.51%，双重 RPA 与 TaqMan 实时 PCR 检测 Em 的符合率为 98.84%，表明其具有准确的临床诊断潜力。因此，本研究建立了一种快速灵敏的双重 RPA 检测方法，可在一个反应管中快速检测和区分 Eg 和 Em，并为现场包虫病检测提供了一种新的检测方法。
