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Abstract
Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.
Highlights
Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis, may be involved (Ansumana et al, 2017; Scott et al, 2017)
To verify the effectiveness of the two sets of primers, we implemented confirmation tests for the singlex Loopmediated isothermal amplification (LAMP) and mLAMP assays using genomic DNA extracted from pure cultures of MTB
The red bands of the control line (CL) and test line 1 (TL1 for IS6110 detection) or test line 2 (TL2 for mtp40 detection) were seen in single LAMP; CL, TL1, and TL2 were red bands in mLAMP experiments, but only CL was shown red in the negative control (Figure 2B)
Summary
Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis, may be involved (Ansumana et al, 2017; Scott et al, 2017). In 2016, approximately 10.4 million new TB cases (most of the cases were MTB infections) were reported globally, of which 147,000 new zoonotic TB cases (mainly M. bovis) accounted for 1.4% of the global incidences of TB (WHO, 2016; Devi et al, 2021). The ability to rapidly detect and differentiate MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB (Spositto et al, 2014). Quick and sensitive diagnostic techniques are required for the detection and differentiation of specific MTBC members in populations
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