Visual detection of Mycobacterium tuberculosis in exhaled breath using N95 enrichment respirator, RPA, and lateral flow assay

Author(s):

Jie Cheng,Yiwei An,Qiyou Wang,Zuanguang Chen,Yanli Tong

Journal:

Talanta

Year:

2025

Abstract:

Tuberculosis (TB) is the second deadliest infectious disease worldwide. Current TB diagnostics utilize sputum samples, which are difficult to obtain, and sample processing is time-consuming and difficult. This study developed an integrated diagnostic platform for the rapid visual detection of Mycobacterium tuberculosis (Mtb) in breath samples at the point-of-care (POC), especially in resource-limited settings. The less pathogenic Mycobacterium smegmatis containing same gene fragment of Mtb served as the model bacterium. A novel respirator was designed to collect airborne mycobacteria in breath samples, with an efficiency of 38.7-61.5 % (102-109 CFU/mL). In our vision, patients only needed to wear a respirator for 1 h, and the collected pathogens were loaded into a microfluidic chip with direct-current electric field for lysis and nucleic acid extraction (20 μL, 3 s), then recombinase polymerase amplification (36 °C, 8 min) and lateral flow strip assay (5 min) were proceeded to enable visual test for the POC. Our platform completed the entire sample collection and diagnosis within 90 min, and the bacterial DNA amplification can be completed in 8 min by handheld, showing great patient compliance and eliminating the need for large equipment. Diagnostic systems involving signal detection with the naked eye are more suitable for the large-scale screening of TB. The proposed method detected low concentrations of bacterial DNA (5.0 aM, 18 copies/μL) with high reproducibility and specificity. Moreover, the system accurately detected low bacterial concentrations (102 CFU/mL). This platform provides the potential to improve the screening of TB and other airborne infectious diseases.