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Journal of Clinical Microbiology, March 2000, p. 1121-1126, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genome-Sequence-Based Fluorescent Amplified-Fragment Length Polymorphism Analysis of Mycobacterium tuberculosis

Jonathan N. Goulding, John Stanley, Nick Saunders, and Catherine Arnold*

Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, London NW9 5HT, United Kingdom

Received 27 September 1999/Returned for modification 7 November 1999/Accepted 11 December 1999

The whole-genome fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP) analysis, was applied to Mycobacterium tuberculosis. Sixty-five clinical isolates were analyzed to determine the value of FAFLP as a stand-alone genotyping technique and to compare it with the well-established IS6110 typing system. The genome sequence of M. tuberculosis strain H37Rv (S. T. Cole et al., Nature 393:537-544, 1998) was used to model computer-generated informative primer combination(s), and the precision and reproducibility of FAFLP were evaluated by comparing the results of in vitro and computer-generated experiments. Multiplex FAFLP was used to increase resolving power in a predictable and systematic fashion. FAFLP analysis was broadly congruent with IS6110 typing for those strains with multiple IS6110 copies. It was also able to resolve an epidemiologically unlinked group of strains with only one copy of IS6110; up to 10% of clinical isolates may fall into this category. For certain epidemiological investigations, it was concluded that a combination of FAFLP and IS6110 typing would give higher resolution than would either alone. FAFLP data were digital, precise, reproducible, and suitable for rapid electronic dissemination, manipulation, interlaboratory comparison, and storage in national or international epidemiological databases. Because FAFLP samples and analyzes base substitution across the genome as a whole, FAFLP could generate new information about the microevolution of the M. tuberculosis complex.

* Corresponding author. Mailing address: Molecular Biology Unit, Virus Reference Division, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: 181 200 4400. Fax: 181 200 1569. E-mail: carnold{at}hgmp.mrc.ac.uk.

Journal of Clinical Microbiology, March 2000, p. 1121-1126, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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