Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis

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...

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Bibliographic Details
Published in:Virchows Archiv : an international journal of pathology 1995-08, Vol.427 (1), p.85-89
Main Authors: TÖTSCH, M, BRÖMMELKAMP, E, STÜCKER, A, FILLE, M, GROSS, R, WIESNER, P, SCHMID, K. W, BÖCKER, W, DOCKHORN-DWORNICZAK, B
Format: Article
Language:English
Subjects:
Algorithms
Bacterial Typing Techniques
Base Sequence
Biological and medical sciences
DNA, Bacterial - analysis
Investigative techniques, diagnostic techniques (general aspects)
Medical sciences
Miscellaneous. Technology
Molecular Sequence Data
Mycobacterium - genetics
Mycobacterium - isolation & purification
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Polymorphism, Restriction Fragment Length
Restriction Mapping
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Summary: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.
ISSN:0945-6317
1432-2307
DOI:10.1007/BF00203742