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

Abstract

<h4>Background</h4>Mycobacterium tuberculosis (MTB) retains infectivity for 8-10 days in dry dust and survives up to 6-8 months in humid environments. Dust accumulated in ventilation systems and air-conditioning filters serves as a critical reservoir for MTB aerosol transmission, making its detection vital for environmental risk assessment in settings prone to clustered outbreaks, such as schools and hospitals. However, molecular detection of MTB in dust is hindered by inhibitors (e.g., humic acids and heavy metals), leading to high false-negative rates, while conventional decontamination methods (e.g., acid-base treatments) compromise bacterial viability and efficiency.<h4>Methods</h4>This study developed a non-destructive Percoll density gradient centrifugation method applied to both H37Rv-spiked artificial dust matrices and real-world air-conditioning dust samples from tuberculosis wards, enabling efficient isolation of viable MTB. This approach leverages buoyant density-based separation to minimize inhibitory effects and maintain bacterial viability.<h4>Results</h4>(1) For H37Rv-spiked artificial dust (10⁰-10⁸ CFU/mL), total recovery rates were 31.75-73.96% with up to 73.5% of recovered bacteria retaining culturability; (2) The method showed no significant discrimination between live and heat-inactivated H37Rv: recovery rates of pure viable and heat-inactivated groups were comparable, and observed recoveries of live/heat-inactivated mixtures mostly matched expected values; (3) For 25 real-world dust samples, qPCR-positive detection rate increased from 56% (direct extraction) to 80%, MTB DNA abundance elevated nearly 10-fold (1,377.73 vs. 143.91 copies/mL), with reduced Ct values, minimized inhibitor interference, and improved amplification efficiency/template quality, enhancing high-risk ward identification.<h4>Conclusion</h4>This method is operationally simple, non-toxic, and biocompatible, achieving high total recovery of MTB cells while maintaining a high proportion of viable among the recovered bacteria. It offers substantial potential for environmental monitoring and downstream sequencing analysis, aiding in environmental risk assessment and management of clustered tuberculosis outbreaks.