Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated...

Area of Science:

• Microbiology
• Biotechnology
• Infectious Diseases

Background:

• Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), remains a leading infectious cause of death globally.
• Early and accurate diagnosis is crucial for effective TB control, particularly in low-income populations.
• Existing diagnostic methods often lack the speed, sensitivity, or cost-effectiveness required for point-of-care use in resource-constrained areas.

Purpose of the Study:

• To develop and validate a novel, rapid, and sensitive nucleic acid detection strategy for MTBC.
• To create a cost-saving point-of-care diagnostic test for early TB detection.
• To establish a multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor (mLAMP-LFB) assay for MTBC identification.

Main Methods:

• Design and validation of two sets of loop-mediated isothermal amplification (LAMP) primers targeting the IS6110 and gyrB genes of MTBC.
• Optimization of reaction conditions (65°C for 40 min) for mLAMP.
• Integration of mLAMP with a nanoparticle-based lateral flow biosensor (LFB) for visual detection of amplification products within 2 minutes.

Main Results:

• The mLAMP-LFB assay successfully detected MTBC with high specificity (100%) and no cross-reactivity.
• The assay demonstrated a limit of detection of 100 fg of DNA in pure culture.
• The entire assay, from DNA extraction to detection, could be completed in approximately 80 minutes.

Conclusions:

• The developed mLAMP-LFB technology offers an objective, rapid, simple, and sensitive method for MTBC identification.
• This assay is suitable for clinical application, especially in resource-limited settings, facilitating early TB diagnosis.
• The findings support the advancement of the WHO's End Tuberculosis Strategy through improved diagnostic capabilities.