Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China; Changping Laboratory, Beijing, China; Laboratory of Clinical Microbiology and Infectious Diseases, Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, National Clinical Research Center for Respiratory Diseases, National Center for Respiratory Medicine, China-Japan Friendship Hospital, Beijing, China; Institute of Respiratory Medicine, Chinese Academy of Medical Sciences, Beijing, China.
Biosens Bioelectron. 2025 Jan 1;267:116740. doi: 10.1016/j.bios.2024.116740. Epub 2024 Sep 5.
Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.
肺炎克雷伯菌对全球公共健康构成重大威胁。传统的临床诊断方法，如细菌培养和显微镜鉴定，不适用于即时检测。针对这一问题，本研究基于非最佳原间隔基序，开发了一种无需提取的一步法检测方法，命名为 EXORCA（EXtraction-free One-pot RPA-CRISPR/Cas12a assay），用于快速、灵敏、高效地检测肺炎克雷伯菌。EXORCA 检测法可在恒温下约 30 分钟内完成，结果可通过荧光读数仪或在蓝光下直接用肉眼观察。该方法用 20 份未经提取的临床样本进行了可行性评估，与 qPCR 相比，其阳性预测值为 100%（5/5），阴性预测值为 100%（15/15）。这些结果表明，EXORCA 检测法有望成为一种即时检测工具，用于快速鉴定病原体，如肺炎克雷伯菌。
