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J Clin Microbiol. Jul 1993; 31(7): 1688–1694.
PMCID: PMC265615

Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.

Abstract

The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 microliters) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 microM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.

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Selected References

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  • Böddinghaus B, Rogall T, Flohr T, Blöcker H, Böttger EC. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. [PMC free article] [PubMed]
  • Brisson-Noël A, Gicquel B, Lecossier D, Lévy-Frébault V, Nassif X, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet. 1989 Nov 4;2(8671):1069–1071. [PubMed]
  • Buck GE, O'Hara LC, Summersgill JT. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol. 1992 May;30(5):1331–1334. [PMC free article] [PubMed]
  • Cousins DV, Wilton SD, Francis BR, Gow BL. Use of polymerase chain reaction for rapid diagnosis of tuberculosis. J Clin Microbiol. 1992 Jan;30(1):255–258. [PMC free article] [PubMed]
  • De Wit D, Steyn L, Shoemaker S, Sogin M. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J Clin Microbiol. 1990 Nov;28(11):2437–2441. [PMC free article] [PubMed]
  • Eisenach KD, Cave MD, Bates JH, Crawford JT. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis. 1990 May;161(5):977–981. [PubMed]
  • Eisenach KD, Sifford MD, Cave MD, Bates JH, Crawford JT. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis. 1991 Nov;144(5):1160–1163. [PubMed]
  • Hermans PW, Schuitema AR, Van Soolingen D, Verstynen CP, Bik EM, Thole JE, Kolk AH, van Embden JD. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J Clin Microbiol. 1990 Jun;28(6):1204–1213. [PMC free article] [PubMed]
  • Hermans PW, van Soolingen D, Dale JW, Schuitema AR, McAdam RA, Catty D, van Embden JD. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol. 1990 Sep;28(9):2051–2058. [PMC free article] [PubMed]
  • Pao CC, Yen TS, You JB, Maa JS, Fiss EH, Chang CH. Detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin Microbiol. 1990 Sep;28(9):1877–1880. [PMC free article] [PubMed]
  • Pierre C, Lecossier D, Boussougant Y, Bocart D, Joly V, Yeni P, Hance AJ. Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol. 1991 Apr;29(4):712–717. [PMC free article] [PubMed]
  • Del Portillo P, Murillo LA, Patarroyo ME. Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol. 1991 Oct;29(10):2163–2168. [PMC free article] [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Sjöbring U, Mecklenburg M, Andersen AB, Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Oct;28(10):2200–2204. [PMC free article] [PubMed]
  • Soini H, Skurnik M, Liippo K, Tala E, Viljanen MK. Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. J Clin Microbiol. 1992 Aug;30(8):2025–2028. [PMC free article] [PubMed]
  • Victor T, du Toit R, van Helden PD. Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction. J Clin Microbiol. 1992 Jun;30(6):1514–1517. [PMC free article] [PubMed]

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