State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
School of Laboratory Medicine and Life Sciences, Wenzhou Medical Universitygrid.268099.c, Wenzhou, China.
Microbiol Spectr. 2022 Aug 31;10(4):e0154522. doi: 10.1128/spectrum.01545-22. Epub 2022 Jul 20.
Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens causing nosocomial infection. A rapid, accurate, and convenient detection method is required for early diagnosis and directed therapy of K. pneumoniae infection. CRISPR-top (CRISPR-mediated testing in one pot) is a LAMP-CRISPR-based nucleic acid detection platform, which integrates target preamplification with CRISPR/Cas12b-based detection into a one-pot reaction mixture, performed at a constant temperature. In this study, we established the K. pneumoniae CRISPR-top assay to precisely identify K. pneumoniae at 56°C within 60 min. The reaction mixture with 0.53 μM (each) FIP and BIP, 0.27 μM LF, 0.13 μM (each) F3 and B3, and 2 μM ssDNA fluorescence probe was determined as the optimal reaction system of our assay. The limit of detection of this assay is 1 pg genomic DNA (equivalent to 160 K. pneumoniae cells and 1.6 × 10 CFU/mL for samples) per reaction, which is 10-fold more sensitive than LAMP. Up to 105 strains composed of K. pneumoniae clinical isolates and non-K. pneumoniae strains were correctly identified by our assay. A total of 58 sputum samples collected from patients with respiratory symptoms were used to evaluate the diagnostic performance of the K. pneumoniae CRISPR-top assay. As a result, the K. pneumoniae CRISPR-top assay yielded 100% (33/33) specificity and 96% (24/25) sensitivity, as well as a positive predictive value of 100% (24/24) and a negative predictive value of 97.1% (33/34), which were all higher than LAMP detection. In conclusion, the K. pneumoniae CRISPR-top assay developed in this study is a simple, rapid and ultra-specific method to detect K. pneumoniae. Klebsiella pneumoniae is a significant threat to global health. At present, the methods of K. pneumoniae detection are culture-based and instrument-dependent and are not suitable for rapid diagnostic. This study reports K. pneumoniae CRISPR-top assay, which can precisely identify K. pneumoniae using nucleic acids of pure cultures or clinical samples in one pot with one fluid-handling step. The K. pneumoniae CRISPR-top reaction can be completed within 60 min at a constant temperature, thus specific instruments are not required. Our results show that CRISPR-top assay yields enormous advantages compared with LAMP detection. The K. pneumoniae CRISPR-top assay can be a high-efficiency alternative tool for rapid and accurate diagnosis of K. pneumoniae infection, especially in resource-limited settings.
肺炎克雷伯菌（K. pneumoniae）是引起医院感染的最常见病原体之一。需要一种快速、准确、方便的检测方法，以便对肺炎克雷伯菌感染进行早期诊断和靶向治疗。CRISPR-top（CRISPR 介导的一锅检测）是一种基于 LAMP-CRISPR 的核酸检测平台，它将靶标预扩增与基于 CRISPR/Cas12b 的检测集成到一个一锅反应混合物中，在恒温下进行。本研究建立了肺炎克雷伯菌 CRISPR-top 检测方法，可在 56°C 下 60 分钟内精确鉴定肺炎克雷伯菌。反应混合物中含有 0.53 μM（各）FIP 和 BIP、0.27 μM LF、0.13 μM（各）F3 和 B3 以及 2 μM ssDNA 荧光探针，被确定为我们检测方法的最佳反应体系。该检测方法的检测限为每个反应 1pg 基因组 DNA（相当于 160 个肺炎克雷伯菌细胞和 1.6×10 CFU/mL 的样品），比 LAMP 灵敏 10 倍。我们的检测方法可正确识别由肺炎克雷伯菌临床分离株和非肺炎克雷伯菌菌株组成的多达 105 株菌株。共使用 58 份来自有呼吸道症状患者的痰液样本评估了肺炎克雷伯菌 CRISPR-top 检测的诊断性能。结果表明，肺炎克雷伯菌 CRISPR-top 检测的特异性为 100%（33/33），敏感性为 96%（24/25），阳性预测值为 100%（24/24），阴性预测值为 97.1%（33/34），均高于 LAMP 检测。总之，本研究建立的肺炎克雷伯菌 CRISPR-top 检测方法是一种简单、快速和超高特异性的检测肺炎克雷伯菌的方法。
肺炎克雷伯菌对全球健康构成重大威胁。目前，肺炎克雷伯菌的检测方法基于培养和仪器，不适合快速诊断。本研究报告了肺炎克雷伯菌 CRISPR-top 检测方法，该方法可以在一个管中通过一步液体处理，使用纯培养物或临床样本的核酸进行一锅式快速鉴定肺炎克雷伯菌。肺炎克雷伯菌 CRISPR-top 反应可以在 60 分钟内恒温完成，因此不需要特定的仪器。我们的结果表明，CRISPR-top 检测与 LAMP 检测相比具有巨大优势。肺炎克雷伯菌 CRISPR-top 检测可能成为一种高效的替代工具，用于快速准确地诊断肺炎克雷伯菌感染，尤其是在资源有限的情况下。
