End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific.

IF 2.3 2区 农林科学 Q1 VETERINARY SCIENCES
Yilin Wang, Fuchang Yu, Kaihui Zhang, Ke Shi, Yuancai Chen, Junqiang Li, Xiaoying Li, Longxian Zhang
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Abstract

Enterocytozoon bieneusi is a common species of microsporidia that infects humans and animals. Current methods for detecting E. bieneusi infections have trade-offs in sensitivity, specificity, simplicity, cost and speed and are thus unacceptable for clinical application. We tested the effectiveness of a previously reported CRISPR/Cas12a-based method (ReCTC) when used for the nucleic acid detection of E. bieneusi. The limit of detection (LOD) and the specificity of the expanded ReCTC were evaluated using prepared target DNA, and the accuracy of the ReCTC-based detection of E. bieneusi in clinical samples was validated. The ReCTC method was successfully used for the nucleic acid detection of E. bieneusi. The sensitivity test indicated an LOD of 3.7 copies/µl for the ReCTC-based fluorescence and lateral flow strip methods. In specificity test involving other common enteric pathogens, a fluorescent signal and/or test line appeared only when the sample was positive for E. bieneusi. These results demonstrated that the ReCTC method can successfully detect E. bieneusi in clinical samples. The ReCTC method was successfully used to detect E. bieneusi nucleic acid with high sensitivity and specificity. It had excellent performance in clinical DNA samples and was superior to nested polymerase chain reaction. Furthermore, the ReCTC method demonstrated its capability for use in on-site detection.

基于端点RPA-CRISPR/ cas12的双氏肠细胞虫核酸检测:快速、敏感、特异。
bieneusenterocytozoon bieneusi是一种常见的小孢子虫,感染人类和动物。目前检测比氏埃希氏菌感染的方法在敏感性、特异性、简单性、成本和速度方面存在权衡,因此不适合临床应用。我们测试了先前报道的基于CRISPR/ cas12的方法(ReCTC)用于bieneusii核酸检测的有效性。利用制备的靶DNA评价扩展ReCTC的检出限(LOD)和特异性,验证了基于ReCTC的临床样品中bieneusi检测的准确性。ReCTC方法可成功地用于比氏伊布氏杆菌的核酸检测。灵敏度试验表明,基于rectc的荧光法和横向流动条法的LOD为3.7 copies/µl。在涉及其他常见肠道病原体的特异性试验中,只有当样品呈比氏埃希菌阳性时才会出现荧光信号和/或检测线。结果表明,ReCTC方法可成功检测出临床样品中的比氏埃希菌。ReCTC方法具有较高的灵敏度和特异度,能成功地检测出比氏鄂氏杆菌核酸。它在临床DNA样品中表现优异,优于巢式聚合酶链反应。此外,ReCTC方法证明了其用于现场检测的能力。
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来源期刊
BMC Veterinary Research
BMC Veterinary Research VETERINARY SCIENCES-
CiteScore
4.80
自引率
3.80%
发文量
420
审稿时长
3-6 weeks
期刊介绍: BMC Veterinary Research is an open access, peer-reviewed journal that considers articles on all aspects of veterinary science and medicine, including the epidemiology, diagnosis, prevention and treatment of medical conditions of domestic, companion, farm and wild animals, as well as the biomedical processes that underlie their health.
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