Duplex recombinase aided amplification-lateral flow dipstick assay for rapid distinction of Mycobacterium tuberculosis and Mycobacterium avium complex.

IF 4.6 2区 医学 Q2 IMMUNOLOGY
Frontiers in Cellular and Infection Microbiology Pub Date : 2024-10-10 eCollection Date: 2024-01-01 DOI:10.3389/fcimb.2024.1454096
Ke Chen, Junze Zhang, Simeng Wang, Zhengjun Yi, Yurong Fu
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引用次数: 0

Abstract

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples.

Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance.

Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi, M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 102 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients.

Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments.

用于快速区分结核分枝杆菌和复合分枝杆菌的双相重组酶辅助扩增-侧流滴定法。
研究目的本研究旨在开发一种新型诊断方法,利用重组酶辅助扩增-侧流点液法(RAA-LFD)区分结核分枝杆菌(MTB)和复合分枝杆菌(MAC),从而在临床样本中快速、便捷、准确地识别这两种分枝杆菌:我们的研究建立了一种能区分 MTB 和 MAC 的双联 RAA-LFD 检测方法。根据 RAA 引物和探针设计原理,分别针对 MTB IS6110 和 MAC DT1 开发了特异性引物和探针。对反应时间点和温度进行了优化,随后对特异性、灵敏度和重现性进行了评估。然后将建立的检测方法应用于临床样本,并与涂片显微镜、液体培养、LAMP 和 Xpert/MTB RIF 的诊断性能进行比较:结果:完整的工作流程可在 37°C 恒温条件下于 20 分钟内有效扩增 MTB IS6110 和 MAC DT1 目标序列,并可在 LFD 检测条上直观观察扩增产物。该方法具有高度特异性,与卡萨斯马立克氏菌、脓肿马立克氏菌、戈登马立克氏菌、螯合马立克氏菌、福氏马立克氏菌、瘰疬马立克氏菌、恶性马立克氏菌、M. chimaera 马立克氏菌、M. szulgai 马立克氏菌和常见呼吸道病原体的核酸无交叉反应。该方法灵敏度高,检测限低至 102 CFU/mL。此外,该方法的变异系数(CV)小于 5%,确保了出色的重复性和可靠性。此外,以 Xpert/MTB RIF 为金标准进行的临床性能评估表明,RAA-LFD 双重检测法的灵敏度为 92.86%,特异性为 93.75%。值得注意的是,该检测方法对涂片阴性患者的诊断效果也相当不错:我们的研究引入了一种快速、特异、灵敏的 RAA-LFD 双重检测法,用于 MTB 和 MAC 的鉴别诊断。这种方法代表了传染病诊断领域的一大进步,为快速检测和管理 MTB 和 MAC 感染提供了有价值的工具。在护理点环境中采用这种方法可大大加强结核病的控制和预防工作,尤其是在资源有限的环境中。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
7.90
自引率
7.00%
发文量
1817
审稿时长
14 weeks
期刊介绍: Frontiers in Cellular and Infection Microbiology is a leading specialty journal, publishing rigorously peer-reviewed research across all pathogenic microorganisms and their interaction with their hosts. Chief Editor Yousef Abu Kwaik, University of Louisville is supported by an outstanding Editorial Board of international experts. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide. Frontiers in Cellular and Infection Microbiology includes research on bacteria, fungi, parasites, viruses, endosymbionts, prions and all microbial pathogens as well as the microbiota and its effect on health and disease in various hosts. The research approaches include molecular microbiology, cellular microbiology, gene regulation, proteomics, signal transduction, pathogenic evolution, genomics, structural biology, and virulence factors as well as model hosts. Areas of research to counteract infectious agents by the host include the host innate and adaptive immune responses as well as metabolic restrictions to various pathogenic microorganisms, vaccine design and development against various pathogenic microorganisms, and the mechanisms of antibiotic resistance and its countermeasures.
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