Helmi M, D, Mohamed Amine S, Besma M, Saloua B F and Rob W
Pasteur Institute of Tunis, Tunisia
Scientific Tracks Abstracts: Arch Clin Microbiol
Several limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the method that served for long as a gold standard for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5’3’FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5’ and 3’ IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5’3’FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility. Here we introduced critical amendments to the original IS6110- 5’3’FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses. IS6110-5’3’FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments, (ii) omission of the plasmid amplification step in E. coli, (iii) the use of more stable fluorophores, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data. In doing so, the overall turnaround time of IS6110-5’3’FP was reduced to 4 hours. The new protocol allowed detecting almost all 5’ and 3’ IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5’3’FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4. In conclusion, the IS6110-5’3’FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRUVNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.
Helmi M, Doctor in Veterinary Medicine (DVM), has carried out his PhD in Virology Immunology at the Institut Armand-Frappier, University of Quebec. He is head of a research laboratory of the Institut Pasteur de Tunis. Currently, Dr Mardassi is leading a research group focusing on the delineation of the molecular mechanisms underlying emergence of TB outbreaks, the success of particular M. tuberculosis clones, and the impact of drug resistance on fitness. He has over 30 publications with an H-index of 19.
E-mail: [email protected]