An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting

Abstract

A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events.

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

“…In northern Malawi and Botswana, on the other hand, histograms showed a normal distribution for the IS 6110 copies. It has been observed elsewhere that high IS 6110 copy strains can be epidemiologically resolved using a smaller set of loci on MIRU-VNTR genotyping panel [24]. Our data therefore suggests that it is possible to quickly establish epidemiological links for this group of strains using a less laborious and high throughput PCR-based technique.…”

Insertion Element IS6110 based characterisation of Nepalese tuberculosis strains into different genetic lineages

“…The remaining 24.4% of the samples belong to the PGG2 and PGG3 groups (Haarlem, LAM, S, X, T-Uganda and T). However, a limitation of this technique is its difficulty to characterise the samples with less than 4-5 copies of IS6110 as seen in the unassigned group (24.4%) in figure 2, which can be overcome by the use of other typing techniques like Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) [20]. The geographical position of Nepal is likely to have influenced this distribution, with a mixture of predominantly Beijing lineage from the North of the Himalayas and the CAS lineage from the south [ Where B-Blue coloured fragment R-Red coloured fragment G-Green coloured fragment and Y-Black/ Yellow coloured fragment seen in the electropherogram.…”

“…The four-dye FAFLP data collected from the different profiles were then recorded and compared with a reference collection of Mtb isolates [19] using BioNumerics software v6.1 (Applied Maths Inc., Belgium). Fragments common to different lineages (defined as being present in >50% of strains in a particular genetic lineage) were recorded for each Nepalese strain and compared with a fully characterised global collection as detailed by Thorne et al [20]. These data were then used to build a dendrogram using the Dice coefficient of similarities to compare the similarity matrix and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) derived cluster analysis with cophenetic correlation for the branch quality.…”

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

“…Several PCRbased modalities targeting IS6110 have been developed thus far, but none has involved a pre-amplification step. [29][30][31][32][33][34][35] This distinctive feature is likely to endow IS6110-5 0 3 0 FP with increased performance in cases where the starting amount of DNA is very low.…”