Optimized Method for Mycobacteria DNA Extraction from Sputum for Isothermal Amplification.

Sputum sample processing for mycobacterial DNA extraction are demanding process to be integrated into isothermal amplification techniques because they often require procedures with number of centrifugation steps and manual interventions. Here, we established simple use of robust mycobacterial DNA extraction protocol from sputum samples suitable for isothermal amplification technique.The DNA extraction efficiency of each protocol was evaluated using Mycovacterium gordonae (M. gordonae) and M. bovis bacillus Calmette-Guerin (BCG) spiked sputum samples. Three mycobacteria lysis methods, sonication, and heating at two different conditions (heating at 100°C for 20 min and 65°C for 30 min) were evaluated. As spin column based was used for DNA purification step, the optimal DNA adsorption protocol was assessed.Of three mycobacteria lysis methods, the difference of cycle threshold (Ct) values between for M. gordonea and BCG ranged between 0.10 to 0.62 and was not considered as appreciable. The thermal lysis method at 65°C for 30 min was selected due to its easier applicability. For DNA adsorption protocol, one-step protocol of adding DNA adsorption solution containing 4.5 M gudanidinium thiocyanate (GuSCN) before thermal lysis and performing DNA purification using spin column showed achieved efficient DNA extraction.DNA extraction by thermal lysis at 65°C with GuSCN followed by spin column DNA purification is simple, relatively fast (less than 50 min) and robust protocol applicable for downstream isothermal amplification.