Solid-Phase Microextraction of DNA from Mycobacteria in Artificial Sputum Samples To Enable Visual Detection Using Isothermal Amplification

Point-of-care (POC) technologies for the detection of pathogens in clinical samples are highly valued due to their speed, ease of use, and cost-effectiveness. Furthermore, they are ideally suited for resource-limited settings where expensive and sophisticated laboratory equipment may not be readily available. In this study, a rapid method based on solid-phase microextraction (SPME) of mycobacterial DNA with subsequent isothermal amplification and visual detection was developed. Direct coupling of the SPME desorption solution (1 M NaCl) to the isothermal reaction system was achieved to circumvent dilution steps and improve detection limits. Using this method, DNA was preconcentrated from lysed mycobacteria in just 2 min, subjected to isothermal multiple-self-matching-initiated amplification (IMSA), and the amplicons were detected visually. With a total analysis times of less than 2 h, the optimized method was capable of extracting and visually detecting mycobacterial DNA from artificial sputum samples containing clinically relevant concentrations of mycobacteria (10 7 colony forming units/mL), demonstrating its potential for future POC applications.