Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick

Abstract

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

Ultrafast, One-Step, Label-Based Biosensor Diagnosis Platform for the Detection of Mycobacterium tuberculosis in Clinical Applications

“…In the current study, single-target positivity occurred in several sputum specimens (namely, IS 6110- or mpb 64-positive), confirming that the development of a multitarget-based assay to avoid missed diagnosis is an essential strategy (Table S4). Although conventional single LAMP-based techniques targeting IS 6110 or 16s RNA sequences have been developed to detect MTB strains in previous publications (e.g., real-time LAMP, single-step LAMP, and LAMP combined with a lateral flow dipstick), − they are prone to missed detection when the sequence is missing in MTB strains . The real-time mLAMP method targeting the IS 6110 and rpoB genes was established in a previous study .…”

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs

“…We designed three-repeat compaction oligos to target regions used to diagnose pathogens that pose a threat to public health especially in low-income regions. These included oligos against M. tuberculosis ( 30 ), HIV ( 31 ), Influenza-A/H1N1 ( 32 ), and a common β-lactamase producing antimicrobial resistance gene ( 33 ) (table S4). As shown in Fig.…”

“…To demonstrate the flexibility and ease of use of our LAMP compaction approach combined with impedance detection, we designed LAMP compaction oligos against multiple pathogens using as a base previously published LAMP designs (30)(31)(32)(33). We designed three-repeat compaction oligos to target regions used to diagnose pathogens that pose a threat to public health especially in lowincome regions.…”

Latest trends in diagnosis of tuberculosis; A Review.

“…38 Loop isothermal amplification PCR does not require prior purification and detects MTB DNA with high sensitivity and specificity and can produce results in less than 2 hours. 39 However, there is still need to make this test easy, cheaper and more efficient to make it competitive against other PCR methods already available. 40 The Xpert MTB/RIF assay is an automated tool integrated with nucleic acid amplification technique(NAAT)platform isolating MTB and its resistant forms.…”