In:
Journal of Clinical Microbiology, American Society for Microbiology, Vol. 58, No. 10 ( 2020-09-22)
Abstract:
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 10 5 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 10 1 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage ( 〉 97% at 5× depth) occurred at 10 4 BCG cells/ml; 〉 91% coverage (1× depth) occurred at 10 3 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.
Type of Medium:
Online Resource
ISSN:
0095-1137
,
1098-660X
DOI:
10.1128/JCM.00670-20
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2020
detail.hit.zdb_id:
1498353-9
SSG:
12
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