Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105 Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

Original publication

DOI

10.1128/jcm.00670-20

Type

Journal article

Journal

Journal of clinical microbiology

Publication Date

22/09/2020

Volume

58

Addresses

Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford, United Kingdom.