title: IS6110-5'3'FP: an automated typing approach for Mycobacterium tuberculosis complex strains simultaneously targeting and resolving IS6110 5' and 3' polymorphisms.
abstract: OBJECTIVE
  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.affiliation: Unit of Typing and Genetics of Mycobacteria, LR11IPT01 Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, 13 Place Pasteur, BP 74, 1002, Tunis-Belvédère, Tunis, Tunisia.
authors.forename: Sara
authors.initials: S
authors.lastname: Thabet
authors.affiliation: Unit of Typing and Genetics of Mycobacteria, LR11IPT01 Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, 13 Place Pasteur, BP 74, 1002, Tunis-Belvédère, Tunis, Tunisia.
authors.forename: Anis
authors.initials: A
authors.lastname: Karboul
authors.affiliation: Unit of Typing and Genetics of Mycobacteria, LR11IPT01 Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, 13 Place Pasteur, BP 74, 1002, Tunis-Belvédère, Tunis, Tunisia.
authors.forename: Neira
authors.initials: N
authors.lastname: Dekhil
authors.affiliation: Unit of Typing and Genetics of Mycobacteria, LR11IPT01 Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, 13 Place Pasteur, BP 74, 1002, Tunis-Belvédère, Tunis, Tunisia. Electronic address: helmi.merdassi@pasteur.rns.tn.
authors.forename: Helmi
authors.initials: H
authors.lastname: Mardassi
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D004251:DNA Transposable Elements; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results
chemical_list: D004251:DNA Transposable Elements
keywords: Capillary electrophoresis; Differential fluorescent labeling; Digital data; IS6110 RFLP; IS6110-5′3′FP; Molecular typing