Non-continuous Percoll density gradient: a method for purifying Mycobacterium tuberculosis from dust.

Area of Science:

• Environmental microbiology
• Molecular diagnostics
• Infectious disease epidemiology

Background:

• Mycobacterium tuberculosis (MTB) persists in dust, acting as a reservoir for transmission in healthcare and educational settings.
• Detecting MTB in dust is crucial for environmental risk assessment but is hindered by inhibitors and methods that compromise bacterial viability.

Purpose of the Study:

• To develop a non-destructive method for isolating viable MTB from dust samples.
• To improve the accuracy and efficiency of molecular detection of MTB in environmental reservoirs.

Main Methods:

• A Percoll density gradient centrifugation technique was developed for isolating viable MTB from artificial and real-world dust matrices.
• The method utilizes buoyant density separation to minimize inhibitor interference and preserve bacterial viability.

Main Results:

• Achieved 31.75-73.96% total recovery of viable MTB from spiked dust, with up to 73.5% retaining culturability.
• Significantly increased qPCR-positive detection rate from 56% to 80% in real-world dust samples.
• Nearly 10-fold increase in MTB DNA abundance detected, with reduced inhibitor interference and improved amplification efficiency.

Conclusions:

• The developed method is simple, non-toxic, and highly effective for recovering viable MTB from dust.
• This technique enhances environmental monitoring capabilities for tuberculosis, supporting risk assessment and outbreak management.
• Offers potential for downstream genomic analysis to understand MTB transmission dynamics.