Abstract
Tuberculosis is still a major threat to global public health. Here, a novel diagnosis assay, termed as multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB), was developed to simultaneously detect IS6110 and IS1081 of Mycobacterium tuberculosis (MTB) in DNA extracted from reference strain H37Rv and clinical samples. The amplification can be finished within 30 min at a fixed temperature (67°C), thus the whole procedure, including rapid template preparation (15 min), isothermal reaction (30 min) and result reporting (2 min), can be completed within 50 min. The limit of detection of multiplex MCDA assay was 10 fg per reaction. By using the multiplex MCDA protocol, cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was not observed. Among clinically diagnosed TB patients, the sensitivity of liquid culture, Xpert MTB/RIF and multiplex MCDA assay was 42.0% (50/119), 49.6% (59/119), and 88.2% (105/119), respectively. Among culture positive samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 86.0% (43/50) and 98.0% (49/50), respectively. Among culture negative samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 23.2% (16/69) and 81.2% (56/69), respectively. The specificity was 100% (60/60) for Xpert MTB/RIF and 98.3% (59/60) for multiplex MCDA. Therefore, the multiplex MCDA assay for MTB detection is rapid, sensitive and easy to use and may be a promising test for early diagnosis of TB.
Highlights
Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is still a major threat to global public health, with an estimated 10 million new cases and 1.3 million deaths in 2017 (World-Health-Organizaion, 2018)
To address the shortcomings posed by Xpert MTB/RIF and TB-loop-mediated isothermal amplification (LAMP), we developed a novel molecular assay based on the before-mentioned multiple cross displacement amplification (MCDA) (Wang et al, 2015)
We firstly report a MCDA combined with the lateral flow biosensors (LFBs) (MCDA-LFB) method for simple, visible and reliable detection of MTB using two target genes (IS6110 and IS1081) and validate its potential clinical application using clinical samples from patients
Summary
Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is still a major threat to global public health, with an estimated 10 million new cases and 1.3 million deaths in 2017 (World-Health-Organizaion, 2018). Conventional MTB detection methods, including sputum smear microscopy and mycobacterial culture, remain the major tools in the TB endemic countries. Xpert MTB/RIF and loop-mediated isothermal amplification (LAMP) are recommended by the WHO as rapid molecular methods for MTB detection (World-Health-Organizaion, 2011, 2016). To address the shortcomings posed by Xpert MTB/RIF and TB-LAMP, we developed a novel molecular assay based on the before-mentioned multiple cross displacement amplification (MCDA) (Wang et al, 2015). Nanoparticle-based lateral flow biosensors (LFBs) have been devised and applied for rapidly, visually, and objectively indicating the MCDA results (Wang et al, 2018b,c). We firstly report a MCDA combined with the LFB (MCDA-LFB) method for simple, visible and reliable detection of MTB using two target genes (IS6110 and IS1081) and validate its potential clinical application using clinical samples from patients
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