Yoshiro Murase, Makiko Hosoya, Yuta Morishige, Yoshiko Shimomura, Miori Nagai, Aki Tamaru, Akiko Takaki, Satoshi Mitarai, on behalf of Japan Tuberculosis Genotyping Group (2023), Yohei Takahashi, Nozomi Kamitaka, Yusuke Yodotani, Masayuki Yajima, Fumitaka Tani, Shinichiro Shibata, Keiko Fujiwara, Kaori Yamamoto, Kentaro Arikawa, Kaori Shimmen, Hiroki Hayashi, Atsuko Tanouchi, Hitoshi Otsuka, Saori Ueda, Yuina Yamaguchi, Akito Mizokoshi
ABSTRACT Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method’s superiority over conventional methods for Mycobacterium tuberculosis (DNA yield: 17.9 vs 1.9 µg, purity A260/A230: 1.86 vs 1.22, both P < 0.001). Single-facility assessment extended these findings to >32 nontuberculous mycobacterial species ( n = 1,058), showing performance comparable to M. tuberculosis ( n = 1,000), with both achieving median yields of 22.2 µg DNA and consistent quality metrics. The chloroform-bead method significantly reduced the processing time from 2 to 3 days to 2 h while ensuring complete sample sterilization, eliminating the need for species-specific optimization. This streamlined and universally applicable protocol represents a practical advancement in mycobacterial DNA extraction methodology, ideal for high-throughput genomic studies and routine clinical diagnostics. IMPORTANCE Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.