Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification—SYBR green I assays

Abstract Background Rapid diagnosis of Mycobacterium tuberculosis ( Mtb ) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification ( RPA ) was developed to detect specific targets of Mtb , IS 6110 and IS 1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye. Method A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria ( NTM ) DNA samples were amplified at IS 6110 and IS 1081 by RPA . After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis ( RPA ‐ AGE ) and SYBR Green I ( RPA ‐S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR . Results The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR , the sensitivities and specificities of RPA ‐ AGE for IS 6110 and IS 1081 were 100%. The specificity of RPA ‐S was 100% for both targets; however, its sensitivities for IS 6110 and IS 1081 were 97.95% and 99.32%, respectively. The limits of detection of IS 6110 RPA ‐ AGE and RPA ‐S were 0.05 and 0.5 ng, respectively, while the LOD s of IS 1081 RPA ‐ AGE and RPA ‐S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross‐reaction with other bacteria. Conclusion A rapid, sensitive, naked eye RPA assay can be integrated into point‐of‐care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.