Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex.

Xingyun Wang,Shuting Quan,Hairong Huang,Weiwei Jiao,Guirong Wang,Adong Shen,Hui Qi,Lin Sun,Yacui Wang,Chen Shen

doi:10.3389/fcimb.2021.666492

Abstract

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (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 IS6110 and IS1081 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.

Highlights

Tuberculosis (TB) is the leading cause of death by single infectious disease worldwide
The Isothermal amplification kit, biotin-14-dATP, biotin-14dCTP were purchased from HuiDexin Biotechnology Co., Ltd (Tianjin, China)
Sample pad, conjugate pad, nitrocellulose membrane (NC) and absorbent pad were purchased from the Jie-Yi Biotechnology

Summary

Introduction

Tuberculosis (TB) is the leading cause of death by single infectious disease worldwide. Smear microscopy is the routine tool for MTBC detection in resource-limited regions for its simplicity and low cost (Steingart et al, 2006). It lacks sensitivity and often requires more than a single visit of a patient to be completed. Mycobacterial culture is still the gold standard for TB diagnosis due to its high sensitivity and specificity. It is time-consuming and technically demanding, which limits its wide application at reference centers. Polymerase chain reaction (PCR)-based techniques (such as conventional PCR, real-time PCR, multiplex PCR) are attractive and promising for detection of MTBC (Lekhak et al, 2016; Kabir et al, 2018; Molaudzi and Molepo, 2019; Nyaruaba et al, 2019), but they need expensive laboratory instruments and complicated technical skill which make them inaccessible in resourcepoor areas

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