Publication Date
2-14-2023
Journal
Microbiology Spectrum
DOI
10.1128/spectrum.02269-22
PMID
36475757
PMCID
PMC9927083
PubMedCentral® Posted Date
12-8-2022
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Humans, Child, BCG Vaccine, Tuberculosis, Tuberculosis, Pulmonary, Mycobacterium bovis, DNA, Bacterial, High-Throughput Nucleotide Sequencing, Mycobacterium tuberculosis, DNA sequencing, extraction, Mycobacterium tuberculosis, stool
Abstract
The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the
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Diseases Commons, Infectious Disease Commons, Medical Cell Biology Commons, Medical Genetics Commons, Medical Immunology Commons
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