Abstract：
Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay\'s performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.
摘要：
囊性包虫病,由于细粒棘球蚴感染，由于缺乏任何已知的有效治疗方法，作为被忽视的热带病提出了重大挑战。主要影响资源不足，远程,和充满冲突的地区，目前检测技术的局限性使这种疾病更加复杂，比如显微镜，物理成像，ELISA,和qPCR，不适合在这些领域应用。CRISPR/Cas12a作为一种有前途的核酸检测工具的出现，以其无与伦比的特异性为特征，灵敏度提高,和快速检测时间，提供了一个潜在的解决方案。在这项研究中,我们提出了一种一锅法CRISPR/Cas12a检测方法，该方法将重组酶聚合酶扩增（RPA）与次优的原型间隔区相邻基序（PAM）和结构化的CRISPRRNA（crRNA）整合在一起，以提高反应效率。使用包虫囊肿掺杂的狗粪便对测定性能进行评估，并对从中国西部不同地区收集的62只狗粪便样品进行检查，证明了其有效性。该测定允许在蓝光下约15分钟的视觉观察测试结果，并且相对于qPCR显示出较高的便携性和反应速度。达到10个拷贝的靶基因标准质粒的敏感性水平。针对四种tape虫物种验证了分析特异性（E.多房性,H.taeniaeformis,贝尼德尼先生,和D.caninum)和另外两个蠕虫(T.犬和肝肝病毒)，Mesocestoidessp.也有阴性结果。这项研究提出了一种快速，敏感,和及时有效的DNA检测方法，用于包虫囊肿和临床狗粪便的颗粒，潜力作为现场检测的替代工具。这种新的测定主要用于诊断细粒大肠杆菌的确定宿主。使用更大的临床粪便样本进行进一步验证是必要的，以及更有效的核酸释放方法的额外探索。
