Extraction of Mycobacterium tuberculosis DNA: a Question of Containment

Abstract

DNA fingerprinting of Mycobacterium tuberculosis by IS6110 restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism, M. tuberculosis remained viable during this process. These data suggest that the extraction of M. tuberculosis DNA should be performed within containment until complete.The standard method employed for DNA fingerprinting of Mycobacterium tuberculosis is IS6110 restriction fragment length polymorphism. This modality requires a large quantity (Ն2 g) of high-molecular-weight DNA (5-7). The DNA extraction protocols currently used for this purpose are based on chemical and enzymatic lysis of the bacterial cells followed by a chloroform-isoamyl alcohol-based DNA extraction, a somewhat lengthy process that raises methodological and biosafety issues. The cultivation of M. tuberculosis requires a biosafety level 3 containment facility and up to 4 weeks of culture time. The extraction necessitates a 2-to 3-day protocol harsh enough to lyse the bacteria yet sufficiently gentle to prevent DNA shearing.Due to the cumbersome nature of extraction protocols, there has been interest in determining at which point M. tuberculosis preparations can be considered inactivated and thus be safely removed from containment. Heating of the culture is widely used, and at least one report has deemed heating at 80°C as sufficient for inactivation (3). However, other reports have raised concerns as to the efficacy of heating (8) and even of further treatment with a combination of lysozyme and proteinase K (2). As such, the question of where and when these preparations may be safely manipulated remains unanswered. It is vital to address this issue, as improper inactivation and premature transfer of extraction mixtures from containment could result in the unnecessary exposure of laboratory personnel to M. tuberculosis.Here, the DNA from clinical M. tuberculosis isolates was extracted using standard protocols (all reagents were from Sigma-Aldrich unless noted). In brief, isolates were inoculated onto Lowenstein-Jensen (L-J) slants, and when luxuriant growth was apparent, all visible colonies were collected into 500 l Tris-EDTA buffer, pH 8.0, and heated for 20 min at 80°C. Lysozyme was then added to each tube (final concentration, 1 mg/ml), followed by incubation at 37°C for 2 h. Ten percent sodium dodecyl sulfate (final concentration, 1.1%) and proteinase K (final concentration, 0.2 mg/ml; Promega Inc.) were then added, and the tubes were vortexed gently and incubated for 20 min at 65°C. A mixture of N-acetyl-N,N,Ntrimethyl ammonium bromide (CTAB; final concentration, 40 mM) and NaCl (final concentration, 0.1 M) was added, followed immediately by the addition of NaCl alone (final concentration, 0.6 M). The tubes were then vortexed until the suspension turned milky and were incubated for 10 min at 65°C. Seven hundred fifty microliters of chloroform-isoamyl alcohol (24:1) was added to each tube, and ...

Rapid Diagnosis of Extensively Drug-Resistant Tuberculosis by Use of a Reverse Line Blot Hybridization Assay

“…All the collected strains were stored at Ϫ70°C. DNA extraction from mycobacterial culture isolates was carried out by the cetyltrimethylammonium bromide-sodium chloride (CTAB-NaCl) method (15).…”

Real Time PCR Assay for the Differentiation of Mycobacterial Species in Bronchial Washings

“…When the growth was detected, Ziehl-Neelsen stain (ZN stain) was carried out in order to confirm the presence of acid fast organisms. DNA was extracted from confirmed acid fast bacilli (AFB) positive isolates according to the standard CTAB (N-Cetyl-N, N, N-trimethyl ammonium bromide) method [7].…”

Rapid Diagnosis of Tuberculous Pleural Effusion by IS6110 Sequence Based PCR

“…[14] Specimens were centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet suspended in 567 μl of TE (Tris EDTA, pH 7.4) buffer, 30 μl 10% SDS (sodium dodecyl sulfate) and 3 μl proteinase K (20 mg/ml), mixed and incubated at 20°C for 1 h. After incubation, 100 μl of 5 M NaCl and 80 μl of high-salt CTAB buffer (containing 4 M NaCl, 1.8% CTAB) were added and mixed followed by incubation at 65°C for 10 min.…”