使用多重引物滚环扩增结合CRISPR/Cas12a进行即时检测。 （原句“Point-of-care testing of in using multiply-primed-RCA coupled with CRISPR/Cas12a.”中“of”和“in”后面缺少具体内容，翻译可能不太准确，建议补充完整句子后再翻译。）
Point-of-care testing of in using multiply-primed-RCA coupled with CRISPR/Cas12a.
作者信息
Sun Yingying, Wu Yaozhou, Wang Yulin, Li Keke, Chang Yanbin, Wei Lianhua
机构信息
School of Public Health, Gansu University of Chinese Medicine, Lanzhou, 730000, PR China.
Department of Clinical Laboratory, Gansu Provincial Hospital, Lanzhou, 730000, PR China.
出版信息
Heliyon. 2024 Sep 7;10(18):e37640. doi: 10.1016/j.heliyon.2024.e37640. eCollection 2024 Sep 30.
PURPOSE
Due to the serious threat of tuberculosis to global health and limitations of existing diagnostic methods, this study combined the CRISPR/Cas12a system with Multiply-primed-RCA (MRCA) technology for Point-of-care Testing (POCT).
METHOD
We utilized T4 and Taq DNA ligases, compared the effects of specific primers and random 6N primers on the method, and integrated MRCA and the CRISPR-Cas12a system in one tube. By optimizing conditions such as the concentration of DNA ligase, the concentration of padlock probes, and the number of cycles, we finally established T4-MRCA-Cas12a and Taq-MRCA-Cas12a methods for both stepwise and one-step.
RESULTS
The limits of detection of the one-step T4/Taq-MRCA-Cas12a were 10aM and 10aM. With no cross-reactivity with DNA from other bacterial strains. The accuracy and specificity were 88 % and 100 % for T4-MRCA-Cas12a, and 96 % and 100 % for Taq-MRCA-Cas12a, respectively.
CONCLUSION
We developed a POCT method that can directly identify MTB through the naked eye.
目的
鉴于结核病对全球健康构成的严重威胁以及现有诊断方法的局限性，本研究将CRISPR/Cas12a系统与多重引物滚环扩增（MRCA）技术相结合用于即时检测（POCT）。
方法
我们利用T4和Taq DNA连接酶，比较了特异性引物和随机6N引物对该方法的影响，并将MRCA与CRISPR-Cas12a系统整合在一管中。通过优化DNA连接酶浓度、锁式探针浓度和循环次数等条件，最终建立了用于逐步法和一步法的T4-MRCA-Cas12a和Taq-MRCA-Cas12a方法。
结果
一步法T4/Taq-MRCA-Cas12a的检测限均为10aM，与其他细菌菌株的DNA无交叉反应。T4-MRCA-Cas12a的准确度和特异性分别为88%和100%，Taq-MRCA-Cas12a的准确度和特异性分别为96%和100%。
结论
我们开发了一种可通过肉眼直接鉴定结核分枝杆菌的即时检测方法。
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