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用于检测细菌污染血样的新型集成等温扩增系统的开发。

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples.

作者信息

Li Jin, Shang Mei-Yun, Deng Shao-Li, Li Min, Su Ning, Ren Xiao-Dong, Sun Xian-Ge, Li Wen-Man, Li Yu-Wei, Li Ruo-Xu, Huang Qing, Lu Wei-Ping

机构信息

Department of Laboratory Medicine, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing, 400042, P.R. China.

出版信息

AMB Express. 2023 Nov 29;13(1):135. doi: 10.1186/s13568-023-01643-7.

DOI:10.1186/s13568-023-01643-7
PMID:38019349
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10686969/
Abstract

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

摘要

由细菌引起的血流感染（BSI）具有高度致病性和致死性，且易发展为全身炎症状态。及时鉴定致病细菌可改善患者预后。传统检测方法不仅耗时，而且此类检测仅限于实验室。重组酶聚合酶扩增结合侧流试纸条（RPA-LFD）在快速核酸检测方面具有很大潜力，但扩增后的开盖操作易污染实验室。因此，建立更有效的一体化等温扩增系统已成为亟待解决的问题。在本研究中，我们设计并制造了一种密封的一体化等温扩增系统。结合该系统，建立并评估了一套用于检测BSI中金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌和流感嗜血杆菌的RPA-LFD检测方法。整个过程可在不到15分钟内完成，结果可用肉眼观察。所开发的RPA-LFD检测方法显示出良好的灵敏度，在七个相似细菌属中未观察到交叉反应。60份临床样本的检测结果表明，所开发的RPA-LFD检测方法在鉴定BSI中的金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌和流感嗜血杆菌方面具有高特异性和灵敏度。总之，我们的结果表明，所开发的RPA-LFD检测方法可替代现有基于PCR的方法，用于基层医院检测BSI中的金黄色葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌和流感嗜血杆菌。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/ca3cc59d4520/13568_2023_1643_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/2db3a9b735d2/13568_2023_1643_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/f5ff0f583721/13568_2023_1643_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/f9ba362072f7/13568_2023_1643_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/ca3cc59d4520/13568_2023_1643_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/2db3a9b735d2/13568_2023_1643_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/f5ff0f583721/13568_2023_1643_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/f9ba362072f7/13568_2023_1643_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d855/10686969/ca3cc59d4520/13568_2023_1643_Fig3_HTML.jpg

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