Outlook for CRISPR-based tuberculosis assays now in their infancy

Huang, Zhen; Zhang, Guoliang; Lyon, Christopher J.; Hu, Tony; Lu, Shan

doi:10.3389/fimmu.2023.1172035

Cited by 4 publications

(1 citation statement)

References 83 publications

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“…Other studies with different methodologies are designed and implicated. Most of them amplified DNA by recombinase polymerase amplification (RPA) or LAMP (Huang et al 2023 ).…”

Section: Assays and Methodsmentioning

confidence: 99%

New insight in molecular detection of Mycobacterium tuberculosis

Mousavi-Sagharchi,

Afrazeh,

Seyyedian-Nikjeh

et al. 2024

AMB Expr

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Mycobacterium tuberculosis, the causative agent of tuberculosis, is a pathogenic bacterium that has claimed millions of lives since the Middle Ages. According to the World Health Organization’s report, tuberculosis ranks among the ten deadliest diseases worldwide. The presence of an extensive array of genes and diverse proteins within the cellular structure of this bacterium has provided us with a potent tool for diagnosis. While the culture method remains the gold standard for tuberculosis diagnosis, it is possible that molecular diagnostic methods, emphasis on the identification of mutation genes (e.g., rpoB and gyrA) and single nucleotide polymorphisms, could offer a safe and reliable alternative. Over the past few decades, as our understanding of molecular genetics has expanded, methods have been developed based on gene expansion and detection. These methods typically commence with DNA amplification through nucleic acid targeted techniques such as polymerase chain reaction. Various molecular compounds and diverse approaches have been employed in molecular assays. In this review, we endeavor to provide an overview of molecular assays for the diagnosis of tuberculosis with their properties (utilization, challenges, and functions). The ultimate goal is to explore the potential of replacing traditional bacterial methods with these advanced molecular diagnostic techniques.

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“…Other studies with different methodologies are designed and implicated. Most of them amplified DNA by recombinase polymerase amplification (RPA) or LAMP (Huang et al 2023 ).…”

Section: Assays and Methodsmentioning

confidence: 99%

New insight in molecular detection of Mycobacterium tuberculosis

Mousavi-Sagharchi,

Afrazeh,

Seyyedian-Nikjeh

et al. 2024

AMB Expr

View full text Add to dashboard Cite

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Dual-mode sensing of Mycobacterium tuberculosis with DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR system

Zhang,

Bai,

Zhang

et al. 2025

Sensors and Actuators B: Chemical

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Rapid and sensitive detection of Mycobacterium tuberculosis using nested multi-enzyme isothermal rapid amplification in a single reaction

Chang,

Zhang,

Liu

et al. 2024

Microbiol Spectr

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Tuberculosis (TB) remains a major global health problem, and there is an urgent need for rapid, sensitive, and easy-to-use diagnostic technologies to improve TB diagnosis. In this study, we developed the nested multi-enzyme isothermal rapid amplification (nestMIRA) assay for TB. We designed several pairs of primers and probes targeting the IS6110 sequence of Mycobacterium tuberculosis (Mtb) and performed combinatorial testing to optimize the performance of the TB nestMIRA assay. The reaction can be performed at a constant temperature of approximately 40°C and completed within 30 minutes in the same tube without opening the central cap. There has been no cross-reactivity with common non-tuberculous mycobacteria (NTB) and respiratory pathogens. The TB nestMIRA assay has a minimum detection limit of 5 copies/μL for H37Rv genomic DNA and a limit of quantification of 100 CFU/ml. To test the diagnostic performance of the TB nestMIRA assay, we conducted a 163-person clinical cohort study using comprehensive reference standards as the gold standard for clinical diagnosis. Our study showed that TB nestMIRA performed slightly better than GeneXpert MTB/RIF (Xpert) (85.27% vs. 82.17%) and significantly better than culture (55.81%) and acid-fast bacillus (AFB) smear (38.76%). The TB nestMIRA assay offers speed, specificity, sensitivity, and convenience. We believe that it has the potential to be a rapid alternative for TB diagnosis, particularly in resource-limited settings. IMPORTANCE In this study, we have successfully developed a method called nested multi-enzyme isothermal rapid amplification (nestMIRA) for the detection of Mycobacterium tuberculosis (Mtb). This method involves a two-step thermostatic amplification process in the same tube and can be read using fluorescence and lateral flow dipstick (LFD) assays. It is known to be rapid, specific, and highly sensitive. Our method has shown promising results in the detection of clinical specimens, and we believe that it can be a valuable tool for the rapid detection of Mtb in a clinical setting.

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Outlook for CRISPR-based tuberculosis assays now in their infancy

Cited by 4 publications

References 83 publications

New insight in molecular detection of Mycobacterium tuberculosis

New insight in molecular detection of Mycobacterium tuberculosis

Dual-mode sensing of Mycobacterium tuberculosis with DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR system

Rapid and sensitive detection of Mycobacterium tuberculosis using nested multi-enzyme isothermal rapid amplification in a single reaction

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