Wang Xingyun, Wang Guirong, Wang Yacui, Quan Shuting, Qi Hui, Sun Lin, Shen Chen, Huang Hairong, Jiao Weiwei, Shen Adong
Key Laboratory of Major Diseases in Children, Ministry of Education, National Key Discipline of Pediatrics (Capital Medical University), National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Beijing, China.
National Tuberculosis Clinical Laboratory, Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing, China.
Front Cell Infect Microbiol. 2021 Apr 27;11:666492. doi: 10.3389/fcimb.2021.666492. eCollection 2021.
The aim of this study was to develop a simple and reliable method to detect (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS and IS sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.
本研究的目的是开发一种简单可靠的方法来检测结核分枝杆菌复合群（MTBC）并初步验证其临床应用。我们开发并评估了一种基于环介导等温扩增法结合侧向流动生物传感器（LAMP-LFB）的检测方法用于检测MTBC。设计了两组靶向MTBC的IS和IS序列的引物，用于建立多重LAMP-LFB检测方法。通过添加异硫氰酸荧光素（FITC）标记引物以及生物素-14-dATP和生物素-14-dCTP，使扩增产物标记上生物素和异硫氰酸荧光素（FITC），并可使用侧向流动生物传感器（LFB）进行可视化检测。确定的多重LAMP-LFB检测的最佳反应条件为66°C反应50分钟。多重LAMP-LFB检测的分析灵敏度为使用纯培养物时基因组模板10 fg，并且未观察到与其他常见细菌和非结核分枝杆菌菌株的交叉反应。总共收集了来自100例结核病患者（62例确诊结核病病例和38例疑似结核病病例）和43例非结核病患者的143份临床样本，用于评估多重LAMP-LFB检测方法的可行性。多重LAMP-LFB检测法（82.0%，82/100）显示出比培养法（47.0%，47/100，P < 0.001）和Xpert MTB/RIF检测法（54.0%，54/100，P < 0.001）更高的灵敏度。重要的是，多重LAMP-LFB检测法检测出另外28例疑似结核病病例，这使得确诊结核病病例的百分比从62.0%（62/100）增加到90.0%（90/100）。多重LAMP-LFB检测法在非结核病患者中的特异性为97.7%（42/43）。因此，多重LAMP-LFB检测法是一种用于检测MTBC的简单、可靠且灵敏的方法，尤其适用于疑似结核病病例和资源有限的环境。
