Development of isothermal amplification assay for the detection of Mycobacterium tuberculosis

Background: World Health Organization (WHO) has incorporated various strategies to strengthen tuberculosis (TB) control programmes. Current diagnostic tools for TB detection require different levels of laboratory sophistication due to the technical complexities, expertise, and biosafety requirement in nucleic acid (NA) based TB testing. It is, therefore, challenging to use NA-based assay in resource-constrained settings. With such limitations, there is a need to develop new approaches that can facilitate point of care (POC) diagnostics. Recombinase polymerase amplification assay (RPA) is an NA-based amplification platform that requires an optimal heat source for accurate diagnosis of a particular disease in a short time. The method is capable of amplifying at low and constant temperature with minimal sample preparation. Methods and materials: In this study, we developed a rapid assay for the detection of Mycobacterium tuberculosis (Mtb) based on the RPA targeting a specific gene. Results: The RPA-based detection of Mtb DNA can be achieved within 20 min at 39 °C. The analytical sensitivity of the test is 1 pg when tested using purified Mtb genomic DNA. The clinical performance of the RPA was evaluated using 177 sputum samples with a culture method as the gold standard. RPA and microscopy were compared to the gold standard in terms of sensitivity (n = 101) and specificity (n = 76). The results showed that the sensitivity and sensitivity of RPA were 94% (95%CI: 89.7, 98.7) and 96% (95%CI: 91.3, 100) respectively, while microscopy had a sensitivity and specificity of 92% (95%CI: 87.3, 97.5) and 94% (95%CI: 92.2, 99.8) respectively. Conclusion: The RPA is shown to be more sensitive and specific compared to microscopy suggesting that the method has the potential to be used as a diagnostic tool for TB in poor resource setting.