Safe Mycobacterium tuberculosis DNA Extraction Method That Does Not Compromise Integrity

Abstract

Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosis without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.The development of an internationally standardized method for the DNA fingerprinting of clinical isolates of Mycobacterium tuberculosis (6) has enabled scientists to gain insight into the disease dynamics of the tuberculosis epidemic in different settings throughout the world. Furthermore, comparison of databases has allowed for the investigation of the global tuberculosis epidemic (2, 7). Since the development of the DNA fingerprinting technique, numerous reports have raised concern about the safety of the DNA extraction procedure (1, 3, 5). These reports have investigated the viability of M. tuberculosis bacilli during different stages of DNA extraction according to the standardized protocol. The findings are contradictory, with certain laboratories reporting that the standardized method is safe (1, 3), while others have reported that the mycobacteria may persist after heat killing and may remain viable even in the interphase during phenol extraction (5). This has raised the concern that the extraction procedure could put scientists at risk of infection (5). To limit such a possibility, it was suggested that the procedure should be conducted under biosafety level 3 conditions (5). Such a step would further complicate an already time-consuming and laborious method and would largely limit DNA fingerprinting to highly specialized laboratories, possibly compromising research in high-burden countries.In our laboratory, we have developed an alternative method for the extraction of high-quality M. tuberculosis DNA for DNA fingerprinting in molecular epidemiological studies. In this procedure, each clinical isolate of M. tuberculosis was inoculated under biosafety level 3 conditions onto two Lowenstein-Jensen (L-J) slants (10 ml L-J medium in 25-ml Bijou bottles) and incubated at 37°C with weekly aeration until confluent growth was observed. Thereafter, the outer surface of each L-J slant bottle was sterilized by swabbing with 5% Hycolin (William Pearson Chemicals, United Kingdom). Twentyfour L-J slants with tightly sealed caps were then placed in a stainless steel rack within an unsealed biosafety autoclave bag (305 by 660 mm; Sterilin, United Kingdom) and transferred to a prewarmed circulating fan oven (580 by 540 by 510 mm) at 80°C for 1 h to ensure heat killing. Three milliliters of extraction buffer (5% sodium glutamate, 50 mM Tris-HCl [pH 7.4], and 25 mM EDTA) was added to each slant in a biosafety class 2 laminar flow hood, and the colonies were gently scraped from the solid medium with a disposable 10-l loop. The suspension from both L-J slants was then transferred to a sterile 50-ml polypropylene Falcon tube containing approximately 20 glass beads (diameter, 4 mm) and vigo...

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