A Novel Rapid Detection Method for Mycobacterium tuberculosis Based on Scattering-Light Turbidity Using Loop-Mediated...

Area of Science:

• Microbiology
• Molecular Diagnostics
• Isothermal Amplification Techniques

Background:

• Accurate detection of Mycobacterium tuberculosis (MTB) is crucial for tuberculosis control.
• Current drug resistance detection methods for MTB lack efficiency and accuracy, and are prone to cross-contamination.
• Existing methods are inadequate for timely clinical decision-making.

Purpose of the Study:

• To develop a rapid nucleic acid detection method for MTB.
• To improve the efficiency, accuracy, and reduce cross-contamination in MTB detection.
• To provide a new technology for rapid clinical diagnosis of tuberculosis.

Main Methods:

• Designed specific primers targeting the MTB-specific gene (Ag85B).
• Employed scattering loop-mediated isothermal amplification (LAMP) for nucleic acid detection.
• Optimized the LAMP reaction system using a self-developed scattering LAMP turbidimeter.

Main Results:

• Optimal reaction conditions determined: 1.5 µL of 100 mmol/L magnesium ions, 3.5 µL of 10 mmol/L dNTPs, 6 µL of 1.6 mol/L betaine, at 65 °C.
• Achieved a minimum detection limit of 12.40 ng/L with a fastest detection time of approximately 10 minutes.
• Demonstrated 100% accuracy in detecting 20 culture-positive samples and no false positives in 20 culture-negative samples, with an average detection time of 24.9 ± 13 minutes.

Conclusions:

• The developed scattering LAMP method offers high detection efficiency and accuracy for MTB.
• The method is cost-effective and significantly reduces the risk of cross-contamination.
• This technique presents a promising new tool for rapid and reliable clinical detection of Mycobacterium tuberculosis.