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Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp.?

ABSTRACT: The insertion sequence IS6110 has an important role in diagnostic PCR and typing of Mycobacterium tuberculosis. We have evaluated a one-tube nested PCR which detects IS6110. Positive results were obtained with DNAs from four of four M. tuberculosis isolates, seven of eight M. fortuitum isolates, seven of seven M. avium-M. intracellulare complex isolates, four of five M. kansasii isolates, four of five M. xenopi isolates, two of four M. malmoense isolates, and one of two M. chelonei isolates. These results were confirmed by hybridization of genomic DNA from bp 505 to 685 of the IS6110 from M. tuberculosis H37Rv. Dot blot hybridization of genomic DNAs from these isolates with the same probe cinfirmed the presence of a homologous sequence in these mycobacterial species. These data suggest that false-positive results may be obtained for clinical samples when some methods based on IS6110 are used [corrected].

SUBMITTER: Kent L

PROVIDER: S-EPMC228396 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundSeveral technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method 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.ObjectivesHere 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.MethodsIS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data.ResultsIn 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.ConclusionsThe IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR 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.

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Demonstration of homology between IS6110 of Mycobacterium tuberculosis and DNAs of other Mycobacterium spp.?

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