Abstract
The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS6110/IS986 ofMycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS6110/IS986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomalMycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to theTaq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purifiedMycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity.
Similar content being viewed by others
References
Bloom BR Back to a frightening future. Nature 1992, 358: 538–539.
Brisson-Noel A, Aznar C, Chureau C, Nguyen S, Pierre C, Bartoli M, Bonete R, Pialoux G, Gicquel B, Garrigue G Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 1991, 338: 364–366.
Telenti A, Marachesi F, Balz M, Bally F, Böttger EC, Bodmer T Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. Journal of Clinical Microbiology 1993, 31: 175–178.
Sjöbring U, Mecklenburg M, Andersen ÅB, Miörner H Polymerase chain reaction for detection ofMycobacterium tuberculosis. Journal of Clinical Microbiology 1990, 28: 2200–2204.
Manjunath N, Shankar P, Rajan L, Bhargava A, Saluja S, Shriniwas Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle 1991, 72: 21–27.
Takewaki S, Okuzumi K, Ishiko H, Nakahara K, Ohkubo A, Nagai R Genus-specific polymerase chain reaction for the mycobacterialdnaJ gene and species-specific oligonucleotide probes. Journal of Clinical Microbiology 1993, 43: 446–450.
Fauville-Dufaux M, Vanfleteren B, De Wit L, Vincke JP, Van Vooren JP, Yates MD, Serruys E, Content J Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85. European Journal of Clinical Microbiology and Infectious Diseases 1992, 11: 797–803.
Soini H, Skurnik M, Liippo K, Tala E, Viljanen M Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. Journal of Clinical Microbiology 1992, 30: 2025–2028.
Eisenach KD, Cave MD, Bates JH, Crawford JT Polymerase chain reaction amplification of a repetitive DNA sequence specific forMycobacterium tuberculosis. Journal of Infectious Diseases 1990, 161: 977–981.
Veringa E, Van Harsselaar B, Hermans P Polymerase chain reaction to detectMycobacterium tuberculosis in a clinical microbiology laboratory. Journal of Microbiological Methods 1992, 16: 139–147.
Kolk AHJ, Schuitema ARJ, Kuijper S, Van Leeuwen J, Hermans PWM, Van Embden JDA, Hartskeerl RA Detection ofMycobacteriurn tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system Journal of Clinical Microbiology 1992, 30: 2567–2575.
Shawar RM, El-Zaatari FAK, Nataraj A, Clarridge JE Detection ofMycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods. Journal of Clinical Methods 1993, 31: 61–65.
Thierry D, Cave MD, Eisenach KD, Crawford JT, Bates JH, Guesdon JL IS6110, an IS-like element ofMycobacteium tuberculosis complex. Nucleic Acids Research 1990, 18: 188.
Hermans PWM, Van Soolingen D, Dale JW, Schuitema ARJ, McAdam RA, Catty D, Van Embden JDA Insertion element IS986 fromMycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. Journal of Clinical Microbiology 1990, 28: 2051–2058.
Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van Dillen PME, Van der Noordaa J Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 1990, 28: 495–503.
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 1977, 2: 95–113.
Sambrook J, Fritsch EF, Maniatis T Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York, 1989.
Young RA, Davis RW Efficient isolation of genes by using antibody probes. Proceedings of the National Academy of Sciences of the USA 1983, 80: 1194–1198.
Baess I, Mansa B Determination of genome size and base ratio on deoxyribonucleic acid from mycobacteria. Acta Pathologica et Microbiologica Scandinavica 1978, 86: 309–312.
Victor T, Du Toit R, Van Helden PD Purification of sputum samples through sucrose improves detection ofMycobacterium tuberculosis by polymerase chain reaction. Journal of Clinical Microbiology 1992, 30: 1514–1517.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Andersen, Å.B., Thybo, S., Godfrey-Faussett, P. et al. Polymerase chain reaction for detection ofMycobacterium tuberculosis in sputum. Eur. J. Clin. Microbiol. Infect. Dis. 12, 922–927 (1993). https://doi.org/10.1007/BF01992166
Issue Date:
DOI: https://doi.org/10.1007/BF01992166