Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus
文献类型： 外文期刊
作者： Huang, Fuqiang 1 ; Li, Xin 1 ; Zhou, Yule 1 ; Tang, Wenqiang 3 ; Dang, Zhisheng 5 ; Kui, Jun 6 ; Zhang, Chunxia 7 ; Zhang, Xu 1 ;
作者机构： 1.Foshan Univ, Sch Life Sci & Engn, Foshan, Peoples R China
2.Nanjing Agr Univ, Coll Vet Med, Nanjing, Peoples R China
3.State Key Lab Hulless Barley & Yak Germplasm Resou, Lhasa, Peoples R China
4.Tibet Acad Agr & Anim Husb Sci, Lhasa, Peoples R China
5.Natl Inst Parasit Dis China CDC, Chinese Ctr Trop Dis Res, WHO Collaborating Ctr Trop Dis, NHC Key Lab Parasite & Vector Biol, Shanghai, Peoples R China
6.Huangzhong Dist Anim Husb & Vet Stn, Xining, Peoples R China
7.Qinghai Agri Anim Husb Vocat Coll, Huangyuan, Anguilla
关键词： Echinococcus granulosus; CRISPR/Cas12a; Detection; POCT; Suboptimal PAM; Suboptimal structure
期刊名称：VETERINARY PARASITOLOGY （ 影响因子：2.0； 五年影响因子：2.4 ）
ISSN： 0304-4017
年卷期： 2024 年 331 卷
页码：
收录情况： SCI
摘要： 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 underresourced, 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 timeefficient 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.
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