Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.
Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.
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耐甲氧西林金黄色葡萄球菌RPA-CRISPR/Cas 12单管快速检测平台
DOI：
10.3390/diagnostics12040829
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发表时间：
2022-03-28
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耐甲氧西林金黄色葡萄球菌（MRSA）是一种严重的健康威胁，在卫生保健环境和社区环境中引起高水平的发病率和死亡率。尽管现有的诊断方法（包括PCR和基于培养的方法）已在临床实践中常规使用，但它们不适合快速即时检测（POCT）。最近，由于CRISPR/Cas技术的发展，出现了快速床旁检测的新可能性。在这项研究中，我们开发了一种快速，准确，无污染的MRSA检测平台，将重组酶聚合酶扩增（RPA）与Cas 12系统整合到一个试管中。基于单管RPA-CRISPR/Cas 12 a平台，通过侧流测试条（LFS）和基于荧光的方法（包括实时和终点荧光）可视化检测结果。该平台允许特异性MRSA检测，荧光法的灵敏度为10个拷贝，LFS的灵敏度范围为10-100个拷贝。对23份MRSA临床分离株的检测结果表明，荧光法和LFS法与qPCR法的符合率分别为100%和95.7%。总之，单管RPA-CRISPR/Cas 12 a平台是一种有效的MRSA检测方法，在未来的实际POCT应用中具有巨大的潜力。
Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10–100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.
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DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harrington LB;Burstein D;Chen JS;Paez-Espino D;Ma E;Witte IP;Cofsky JC;Kyrpides NC;Banfield JF;Doudna JA
通讯作者：
Doudna JA
CRISPR-Cas12a辅助核酸检测
影响因子：
33.5
作者：
Li SY;Cheng QX;Wang JM;Li XY;Zhang ZL;Gao S;Cao RB;Zhao GP;Wang J
通讯作者：
Wang J
影响因子：
5.6
作者：
Ding R;Long J;Yuan M;Zheng X;Shen Y;Jin Y;Yang H;Li H;Chen S;Duan G
通讯作者：
Duan G
影响因子：
8.8
作者：
Liu, Hua;Wang, Jinbin;Tang, Xueming
通讯作者：
Tang, Xueming
影响因子：
11.8
作者：
Stryjewski, Martin E.;Corey, G. Ralph
通讯作者：
Corey, G. Ralph