Abstract
An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of ±1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin-embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guérin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.
Similar content being viewed by others
References
Brenner S, Livak KJ (1989) DNA fingerprinting by sampled sequencing. Proc Natl Acad Sci USA 86:8902–8906
Butler WR, Jost KCJ, Kilburn JO (1991) Identification of mycobacteria by high-performance liquid chromatography. J Clin Microbiol 29:2468–2472
Cawkwell L, Bell SM, Lewis FA, Dixon MF, Taylor GR, Quirke P (1993) Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology. Br J Cancer 67:1262–1267
Dockhorn-Dworniczak B, Schröder S, Dantcheva R, Tötsch M, Stücker A, Brömmelkamp E, Bankfalvi A, Böcker W, Schmid KW (1993) The role of p53 Tumor Suppressor Gene in human thyroid cancer. Exp Clin Endocrinol 101:39–46
Foss RD, Guha-Thakurta N, Conran M, Gutman P (1994) Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Diagn Mol Pathol 3:148–155
Goldmann D, Merril CR (1982) Silver staining of DNA in polyacrylamide gels: linearity and effect of fragment size. Electrophoresis 3:24–26
Greer CE, Peterson SL, Kiviat NB, Manos MM (1991) PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. Am J Clin Pathol 95:117–124
Guo C, Marynen P, Cassiman JJ (1993) A rapid, semiautomated method for Apolipoprotein E genotyping. PCR Methods Appl 2:348–350
Hammond HA, Jin L, Zhong Y, Caskey CT, Chakraborty R (1994) Evaluation of 13 Short Tandem Repeat loci for use in personal identification applications. Am J Hum Genet 55:175–189
Hance AJ, Grandchamp B, Levy-Frebault V, Lecossier D, Rauzier J, Bocart D, Gicquel B (1989) Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol Microbiol 3:843–849
Kessler C, Holtke HJ (1986) Specificity of restriction endonucleases and methylases — a review. Gene 47:1–153
Kohne DE (1990) The use of DNA probes to detect and identify microorganisms. Adv Exp Med Biol 263:11–35
Mansfeld ES, Kronick MN (1993) Alternative labeling techniques for automated fluorescence based analysis of PCR products. Biotechniques 15:274–279
Mayrand PE, Corcoran KP, Ziegle JS, Robertson JM, Hoff LB, Kronick MN (1992) The use of fluorescence detection and internal lane standards to size PCR products automatically. Appl Theor Electrophor 3:1–11
Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol 155:335–350
Musial CE, Tice LS, Stockman L, Roberts GD (1988) Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex. J Clin Microbiol 26:2120–2123
Pao CC, Lin SS, Wu SY, Juang WM, Chang CH, Lin JY (1988) The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes. Tubercle 69:27–36
Plikaytis BB, Plikaytis BD, Yakrus MA, Butler WR, Woodley CL, Silcox VA, Shinnick TM (1992) Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis. J Clin Microbiol 30:1815–1822
Plikaytis BD, Plikaytis BB, Shinnick TM (1992) Computer-assisted pattern recognition model for the identification of slowly growing mycobacteria including Mycobacterium tuberculosis. J Gen Microbiol 138:2265–2273
Rogall T, Flohr T, Bottger EC (1990) Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol 136:1915–1920
Shinnick TM (1987) The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol 169:1080–1088
Smith HO, Birnstiel ML (1976) A simple method for DNA restriction site mapping. Nucleic Acids Res 3:3287–3298
Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T (1993) Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 31:175–178
Tötsch M, Schmid KW, Brömmelkamp B, Stücker A, Puelacher C, Sidoroff G, Mikuz G, Böcker W, Dockhorn-Dworniczak B (1994) Rapid detection of mycobacterial DNA in clinical samples by multiplex-PCR. Diagn Mol Pathol 3:260–264
Tötsch M, Dockhorn-Dworniczak B, Brömmelkamp E, Öfner D, Kreczy A, Fille M, Böcker W, Schmid KW (in press) Diagnostic value of different PCR assays for the detection of mycobacterial DNA in granulomatous lymphadenopathy. J Pathol
Voss H, Schwager C, Wirkner U, Sproat B, Zimmermann J, Rosenthal A, Erfle H, Stegemann J, Ansorge W (1989) Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA. Nucleic Acids Res 17:2517–2527
Wiesner P, Jäger B (1993) Automated screening of DNA Sequence Polymorphisms and confirmation by DNA sequencing (abstract). Human Genome Mapping Workshop:S85
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tötsch, M., Brömmelkamp, E., Stücker, A. et al. Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis. Vichows Archiv A Pathol Anat 427, 85–89 (1995). https://doi.org/10.1007/BF00203742
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00203742