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Tuberculosis (Edinb). 2011 Dec;91 Suppl 1:S43-8. doi: 10.1016/j.tube.2011.10.009. Epub 2011 Nov 15.

Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using Taqman assays.

Author information

1
Division of Epidemiology, School of Public Health in Brownsville, University of Texas Health Science Center at Houston, 80 Fort Brown, SPH Bldg, Brownsville, Texas 78520, USA.

Abstract

Rapid tuberculosis (TB) detection is critical for disease control, and further quantitation of Mycobacterium tuberculosis (Mtb) in sputum is valuable for epidemiological and clinical studies. We evaluated a simple, robust and cost-efficient in-house DNA extraction and downstream Taqman approach for detection and quantitation of Mtb genomes from sputum of newly-diagnosed TB patients and non-TB controls. DNA was extracted using guanidine isothiocyanate and silica-based spin columns in less than 2 h, stored frozen, and Taqman assays were used to detect Mtb with IS6110 and quantify it targeting RD1 and IS1081. The Taqmans had a sensitivity >95% in 108 culture-confirmed TB patients and specificity of 100% in 43 non-TB controls. Genome counts were correlated with the Mycobacterial Growth Indicator Tubes' (MGIT) time-to-detection values (1/TTD × 1000; rho = 0.66; p < 0.001) in 91 TB patients (33 excluded with MGIT contamination). This linear relationship was nearly identical between mycobacteria isolated from sputum and H37Rv Mtb grown in-vitro to its log phase. TB treatment between 3 and 7 days was associated with lower 1/TTD × 1000 values but not with genome counts. Together, our protocol provides rapid, specific, inexpensive and quantitative detection of Mtb DNA in fresh or stored sputa making it a robust tool for prompt TB diagnosis, and with potential use for clinical and epidemiologic studies.

KEYWORDS:

PCR inhibitors; diagnosis; quantitation; real-time quantitative PCR; sputum; tuberculosis

PMID:
22088321
PMCID:
PMC3248966
DOI:
10.1016/j.tube.2011.10.009
[Indexed for MEDLINE]
Free PMC Article

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