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

Thabet, Sara; Karboul, Anis; Dekhil, Naira; Mardassi, Helmi

doi:10.1016/j.ijid.2014.10.004

Cited by 7 publications

(7 citation statements)

References 34 publications

(29 reference statements)

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“…A number of methods have been used for mapping IS 6110 distribution. Some of these are electrophoresis based IS 6110 -RFLP 36 , PCR based fluorescent amplified fragment length polymorphism (fAFLP) 24 , IS 6110 5′ and 3′ fluorescent polymorphism (IS 6110 -5′3′FP) 37 , DNA microarray based SiteMapping 38 and targeted amplification of IS elements followed by sequencing or IS-seq 39 . All these methods have been useful in identification of many IS insertion sites.…”

mentioning

confidence: 99%

Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis

Roychowdhury

Mandal

Bhattacharya

2015

Sci Rep

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Insertion sequence (IS) 6110 is found at multiple sites in the Mycobacterium tuberculosis genome and displays a high degree of polymorphism with respect to copy number and insertion sites. Therefore, IS6110 is considered to be a useful molecular marker for diagnosis and strain typing of M. tuberculosis. Generally IS6110 elements are identified using experimental methods, useful for analysis of a limited number of isolates. Since short read genome sequences generated using next-generation sequencing (NGS) platforms are available for a large number of isolates, a computational pipeline for identification of IS6110 elements from these datasets was developed. This study shows results from analysis of NGS data of 1377 M. tuberculosis isolates. These isolates represent all seven major global lineages of M. tuberculosis. Lineage specific copy number patterns and preferential insertion regions were observed. Intra-lineage differences were further analyzed for identifying spoligotype specific variations. Copy number distribution and preferential locations of IS6110 in different lineages imply independent evolution of IS6110, governed mainly through ancestral insertion, fitness (gene truncation, promoter activity) and recombinational loss of some copies. A phylogenetic tree based on IS6110 insertion data of different isolates was constructed in order to understand genome level variations of different markers across different lineages.

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mentioning

confidence: 99%

Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis

Roychowdhury

Mandal

Bhattacharya

2015

Sci Rep

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“…Aside from using a frequently cutting enzyme, the use of more stable fluorophores could have significantly contributed to the increased sensitivity of IS 6110 -5’3’FP in terms of fragments detection. In its original version, we noticed that 5’-end fragments were much less frequently detected than 3’-end fragments (34.3% vs 65.7%), a finding that was attributed to the relative instability of JOE dye [ 11 ]. Here we replaced JOE with ATTO 532, a fluorophore with higher stability and better spectral properties.…”

Section: Resultsmentioning

confidence: 99%

“…Because IS 6110 RFLP targets only the 3’ polymorphism, the number of peaks expected to be detected by IS 6110 -5’3’FP should be at least twice the number of IS 6110 RFLP bands (5’ and 3’ polymorphic ends). Reproducibility assay was performed with a well characterized IS 6110 RFLP 11-banded clinical strain [ 12 , 13 ], considered herein to as the laboratory reference strain, and which has been previously used in the optimization steps of the original IS 6110 -5’3’FP protocol [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

“…Towards this end, we reconsidered IS 6110 -5’3’FP, a typing approach that we have previously developed [ 11 ], and which we believe could represent a valuable alternative to 24-loci MIRU-VNTR. IS 6110 -5’3’FP relies on a single PCR amplification reaction, coupled to simultaneous differential labeling of 5’ and 3’ IS 6110 polymorphic ends, and their automated fractionation on a capillary sequencer.…”

Section: Introductionmentioning

confidence: 99%

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Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility

et al. 2018

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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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“…IS6110 PCR is a rapid technique (Thabet et al . ) and the most sensitive and specific for M. tuberculosis diagnosis.…”

Section: Current Pulmonary Tuberculosis Diagnostic Testsmentioning

confidence: 99%

Mass spectrometry applied to the identification ofMycobacterium tuberculosisand biomarker discovery

et al. 2016

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Summary An adequate and effective tuberculosis (TB) diagnosis system has been identified by the World Health Organization as a priority in the fight against this disease. Over the years, several methods have been developed to identify the bacillus, but bacterial culture remains one of the most affordable methods for most countries. For rapid and accurate identification, however, it is more feasible to implement molecular techniques, taking advantage of the availability of public databases containing protein sequences. Mass spectrometry (MS) has become an interesting technique for the identification of TB. Here, we review some of the most widely employed methods for identifying Mycobacterium tuberculosis and present an update on MS applied for the identification of mycobacterial species.

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

Abstract: 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.

Cited by 7 publications

References 34 publications

Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis

Analysis of IS6110 insertion sites provide a glimpse into genome evolution of Mycobacterium tuberculosis

Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility

Mass spectrometry applied to the identification ofMycobacterium tuberculosisand biomarker discovery

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