Stephen Carleton, PhD
Research Assistant Professor
Department of Cell Biology
Tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), one of the deadliest pathogens in human history. TB control today relies on case identification and prophylactic treatment with drugs. Although a presumptive diagnosis of tuberculosis can be made based on patient history, clinical and radiological findings, and the presence of acid-fast bacilli in direct smears, the definitive diagnosis of TB relies on the culture and/or molecular genetic identification of acid-fast bacilli. This need for rapid laboratory identification of Mtb has led to the development of amplification-based strain typing assays applied directly to Mtb bacterial DNA or RNA extracted from patient sputum. While identification of Mtb is important for diagnosis, species identification alone yields no epidemiological information about the source of the infection, or it's course through a population. Strain typing allows the tracing of epidemiologically related cases including virulent or MDR strains, identification of nosocomial infections, differentiation of new cases from relapses, and identification of laboratory contaminants.
We have developed a PCR fingerprinting assay that uses outward directed primers homologous to the ends of IS6110. This assay generates distinct and reproducible fingerprints from different Mtb strains, which are fluorescently labeled and analysed using automated methods. We have also determined that Mtb isolates representing a broad sampling of RFLP-defined clusters ("families") could be distinguished by PCR fingerprinting. Genescan analysis of of over 300 reactions reveals that each PCR fingerprint is composed of 35 to 45 distinguishable bands and that all PCR fingerprints exhibit the 154, 214, and 396 bp signature bands described previously and observed in all Mtb fingerprints examined to date. These signature bands are generated also from M. bovis DNA but not from M. avium or M. xenopi. In addition to the signature bands, at least 27 additional bands ranging in size from 130-820 bp occur in most PCR fingerprints. These have been termed "common bands" and are non-descriminatory.
In addition to the signature and common bands, most fingerprints also contain one or more bands that do not correspond to any of these bands and have proved to be discriminatory. For example, the HJ representative strain contains discriminatory bands of 136,159, and 366 bp, not seen in the fingerprints of any other family strains. This strain also contains all 27 common bands, one band of 464 bp which is relatively bright. Similarly, fingerprints from the BA and BA10 family strains each contain a unique band of 126, 130, and 197 bp as well as all 27 common bands. In addition, many strains contain single bands of distinct size, including BF22 (700 bp), LE4 (602 bp), AX15 (200, 323 bp), AI55 (531 bp), and AH1 (intense 303 and unique 310 bp), (Fig. 1B, lanes 4, 10, 8, 11, and 12, respectively.) However, fingerprints from four family strains (001, B1, AX15, AH1, and D30) share one unique band of 252 bp.
- Olubunui Adeosun, Student
- Judy Dong, Pre-doctoral Trainee
- Alex Kougentokas, Intel Volunteer
- Donald Morgenstern, Technical Specialist
- Wei Quan, Institutional Support Specialist
- Harold Neimark, Associate Professor of Microbiology and Immunology
- Carleton, S., The Polymerase Chain Reaction: Principles and Applications. 1992. In: Advances in Genome Biology. JAI Press Inc. 1:175-215.
- Carleton, S., The Polymerase Chain Reaction: Applications in Genomic Analysis. In: Human Chromosomes: Manual of Basic Techniques (second edition).1995 Verma and Babu, eds. p. 312-344.
Peer Reviewed Research Publications:
- Goswami, S. P. Qasba, S. Ghatpande, S. Carleton, A.K. Deshpande, M. Baig, and M.A.Q. Siddiqui. 1994. Differential expression of myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis. Mol. Cell. Biol 14:5130-5138.
- Rushbrook, J.I., C. Weiss, K. Ko, M.H. Feuerman, S. Carleton, A. Ing, and J. Jacoby. 1994. Identification of Alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit. J. Muscle Res. Cell Motility. 15:505-516.
- Doud, S.K., L.-X. Pan, S. Carleton, S. Marmorstein, and M.A.Q. Siddiqui. 1995. Adaptional response in transcription factors during development of myocardial hypertrophy. J. Mol. Cell. Cardiol. 27:2359-2372.
- Neimark, H., M.A. Baig, and S. Carleton. 1996. Direct identifiction and typing of Mycobacterium tuberculosis by PCR. J. Clin. Microbiol. 34:2454-2459.