IS6110-5'3'FP: an automated typing approach for Mycobacterium tuberculosis complex strains simultaneously targeting and resolving IS6110 5' and 3' polymorphisms.

Abstract

OBJECTIVES:

Fingerprinting of Mycobacterium tuberculosis complex strains based on the IS6110 insertion sequence would considerably gain in terms of discriminatory power and versatility if both 5' and 3' polymorphisms were simultaneously targeted, and if it benefited from automated capillary electrophoresis. In response to these requirements, we developed IS6110-5'3'FP (IS6110 5' and 3' fluorescent polymorphisms).

METHODS:

IS6110-5'3'FP involves the construction of an M. tuberculosis genomic library in a plasmid vector using HincII endonuclease, which cuts within the IS6110 sequence. After amplification in Escherichia coli, the library is subjected to selective and simultaneous PCR amplification of IS6110 5' and 3' polymorphic fragments, using differentially labeled fluorescent primers. The resulting amplicons are then fractionated on a capillary sequencer and the signal peaks analyzed as digital data.

RESULTS:

IS6110-5'3'FP consistently detected and resolved both 5' and 3' IS6110 polymorphic fragments (35% and 65%, respectively) with a high level of reproducibility. The method differentiated all M. tuberculosis strains, as did IS6110 restriction fragment length polymorphism (RFLP), the gold standard of IS6110-based typing. Strikingly, the potential of IS6110-5'3'FP to resolve more polymorphic fragments than IS6110 RFLP was demonstrated.

CONCLUSIONS:

IS6110-5'3'FP demonstrated sufficient potential to be a promising automated alternative to IS6110 RFLP, amenable to high throughput analysis and inter-laboratory comparison.

• Authors: Sara Thabet, Anis Karboul, Neira Dekhil, Helmi Mardassi
• Journal: International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases (Int J Infect Dis) Vol. 29 Pg. 211-8 (Dec 2014) ISSN: 1878-3511 [Electronic] Canada
• PMID: 25447727 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chemical References

• DNA Transposable Elements

Topics

• Bacterial Typing Techniques (methods)
• DNA Transposable Elements
• Humans
• Mycobacterium tuberculosis (classification, genetics, isolation & purification)
• Polymerase Chain Reaction
• Polymorphism, Genetic
• Polymorphism, Restriction Fragment Length
• Reproducibility of Results