title: Rapid DNA extraction for specific detection and quantitation of Mycobacterium tuberculosis DNA in sputum specimens using Taqman assays.
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.
authors.affiliation: 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.
authors.forename: Diana I
authors.initials: DI
authors.lastname: Gomez
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Mullin
authors.forename: Francisco
authors.initials: F
authors.lastname: Mora-Guzmán
authors.forename: J Gonzalo
authors.initials: JG
authors.lastname: Crespo-Solis
authors.forename: Susan P
authors.initials: SP
authors.lastname: Fisher-Hoch
authors.forename: Joseph B
authors.initials: JB
authors.lastname: McCormick
authors.forename: Blanca I
authors.initials: BI
authors.lastname: Restrepo
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005260:Female; D016680:Genome, Bacterial; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D004269:DNA, Bacterial
references.citation: Clin Microbiol Rev. 2006 Jan;19(1):165-256
references.pmid: 16418529
references.citation: Appl Environ Microbiol. 2009 Jan;75(2):414-8
references.pmid: 19047396
references.citation: Eur Respir J. 2005 Aug;26(2):339-50
references.pmid: 16055883
references.citation: Int J Tuberc Lung Dis. 2008 Jul;12(7):792-8
references.pmid: 18544206
references.citation: J Bacteriol. 1956 Aug;72(2):132-41
references.pmid: 13366889
references.citation: J Clin Microbiol. 2011 Mar;49(3):797-801
references.pmid: 21177901
references.citation: J Clin Microbiol. 2001 Oct;39(10):3750-2
references.pmid: 11574609
references.citation: J Clin Microbiol. 2007 Dec;45(12):4064-6
references.pmid: 17928422
references.citation: Lancet. 2011 Apr 30;377(9776):1495-505
references.pmid: 21507477
references.citation: J Bacteriol. 1996 Mar;178(5):1274-82
references.pmid: 8631702
references.citation: J Clin Microbiol. 2004 May;42(5):2321-5
references.pmid: 15131224
references.citation: Appl Environ Microbiol. 2008 Jun;74(12):3839-48
references.pmid: 18441113
references.citation: Int J Tuberc Lung Dis. 2010 May;14(5):560-70
references.pmid: 20392348
references.citation: J Clin Microbiol. 1991 Nov;29(11):2468-72
references.pmid: 1774251
references.citation: Clin Infect Dis. 1998 Mar;26(3):775-6
references.pmid: 9524873
references.citation: Infect Immun. 1998 Feb;66(2):794-9
references.pmid: 9453643
references.citation: Am J Respir Crit Care Med. 2005 Nov 1;172(9):1161-8
references.pmid: 16081545
references.citation: J Microbiol Methods. 2006 Nov;67(2):220-9
references.pmid: 16678925
references.citation: Tuberculosis (Edinb). 2011 May;91(3):257-9
references.pmid: 21353641
references.citation: Appl Environ Microbiol. 2004 Aug;70(8):4561-8
references.pmid: 15294786
references.citation: PLoS One. 2008 Feb 06;3(2):e1536
references.pmid: 18253484
references.citation: J Clin Microbiol. 2004 Sep;42(9):4307-9
references.pmid: 15365029
references.citation: J Clin Microbiol. 2005 Dec;43(12):5835-41
references.pmid: 16333064
references.citation: Diagn Microbiol Infect Dis. 2006 Feb;54(2):121-6
references.pmid: 16406184
references.citation: J Clin Microbiol. 1995 Mar;33(3):629-30
references.pmid: 7751368
references.citation: Infect Control Hosp Epidemiol. 2003 Nov;24(11):831-8
references.pmid: 14649771
references.citation: BMC Microbiol. 2005 Oct 03;5:55
references.pmid: 16202138
references.citation: Bull World Health Organ. 2011 May 1;89(5):352-9
references.pmid: 21556303
references.citation: J Immunol. 1990 Apr 1;144(7):2771-80
references.pmid: 2108212
references.citation: Int J Tuberc Lung Dis. 2001 Jun;5(6):569-74
references.pmid: 11409586