Direct detection of unamplified DNA from Mycobacterium tuberculosis using modified gold nanoparticles

Abstract

Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis, MTB), remains a significant global health threat, especially in resource-limited settings. The absence of rapid, affordable, and clinically useful diagnostic methods poses a significant obstacle to global TB elimination efforts. Standard diagnostic methods are either time-consuming or dependent on sophisticated laboratory infrastructure.

Materials and Methods

This study developed and optimized a novel gold nanoparticle (AuNP)-based colorimetric biosensor for the direct, amplification-free detection of M. tuberculosis using a thiolate DNA probe targeting the IS6110 sequence. A key innovation was a simple freeze-thaw lysis protocol that generated crude DNA lysates directly from clinical respiratory samples, thereby eliminating the need for commercial DNA extraction kits. Sixty clinical specimens were analyzed using both PCR-amplified DNA and direct crude extracts. Detection was based on acid-induced AuNP aggregation, producing a visible color change within 15 minutes.

Results

The biosensor achieved a detection limit (LOD) of 0.031 ng µL^-1 of genomic DNA. The method showed 100 % diagnostic sensitivity (95 % CI: [91.2 %-100 %]) and 100 % specificity (95 % CI: [83.2 %-100 %]), entirely consistent with the results from gold standard techniques like culture and GeneXpert. Crucially, the assay successfully identified all forty confirmed positive samples, including smear-negative cases.

Conclusion

By overcoming the main limitations of existing methods, this study presents a rapid, accurate, and amplification-free approach for tuberculosis. This platform has the potential to transform screening and tuberculosis disease control due to its simplicity, affordability, and minimal equipment requirements, making it an effective point-of-care diagnostic tool, particularly in high-risk, low-resource settings.