Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

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.

A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus

“…Among them, polymerase chain reaction (PCR) and PCR-based assay (i.e., multiplex PCR and real-time PCR) are used as the conventional molecular detection methods in clinical examinations of brucellosis ( Hinić et al, 2008 ; Surucuoglu et al, 2009 ; Kang et al, 2011 ). Despite these methods own outstanding analytical capabilities, the shortcomings (special apparatus’ requirements, poor availability, and long detection procedure) restrict their application in the point-of-care and field laboratories ( Li et al, 2019a ; Yang et al, 2021 ). To address the shortcomings of PCR-based techniques, loop-mediated isothermal amplification (LAMP), which includes 4 core primers (F3, B3, FIP, and BIP) and/or two loop primers (LF and LB) on target sequences, was developed in 2000 by Notomi et al (2000) .…”

“…To address the shortcomings of PCR-based techniques, loop-mediated isothermal amplification (LAMP), which includes 4 core primers (F3, B3, FIP, and BIP) and/or two loop primers (LF and LB) on target sequences, was developed in 2000 by Notomi et al (2000) . LAMP technique, as a fast, reliable, simple, and sensitive isothermal detection method, has been performed to detect various pathogens (containing bacteria, viruses, fungi, and emerging/re-emerging infectious agents) ( Wang et al, 2017 ; Kashir and Yaqinuddin, 2020 ; Yang et al, 2021 ). In previous reports, the LAMP technique has been used to detect Brucella ssp .…”

“…in diagnosis assay. Unfortunately, conventional validation methods for LAMP amplicons, including hydroxy naphthol blue (HNB), SYBR Green, and agarose gel electrophoresis, are difficult to accurately distinguish specific amplification from non-specific amplification, which can easily lead to misinterpretation of the results ( Li et al, 2019a ; Yang et al, 2021 ).…”

“…To overcome these defects, a target-specific, simple, and visual nanoparticle-based lateral flow immunoassay biosensor (LFIA) was successfully designed and applied to verify LAMP products ( Li et al, 2019a ; Yang et al, 2021 ). The detection mechanism of LFIA is that LAMP reaction amplicons, which are composed of 6-carboxyfluorescein (6-FAM) and biotin, combined with dye streptavidin-coated polymer nanoparticles (SA-PNPs) to form a complex (6-FAM-biotin-SA-PNPs).…”

“…Although LAMP- and MCDA-LFIA (multiple cross displacement amplification) methods targeting Bscp31 , mcr-1 , IS6110 , and mtp40 gene for detection of Brucella spp. (genus level) and/or other target pathogens have been established, these methods cannot accurately detect and/or identify the Brucella abortus strains (species level) ( Li et al, 2019a ; Li et al, 2019b ; Gong et al, 2019 ; Yang et al, 2021 ). Previously, conventional PCR and LAMP assays targeting BruAb2_0168 gene were developed and applied to identify the B. abortus in accurate diagnosis tests of brucellosis ( Hinić et al, 2008 ; Kang et al, 2015 ).…”