Moganeradj, Kartykayan; (2018) IS6110 FAFLP PCR, a tool for genomic mapping enabling investigation of evolutionary relationships of Mycobacterium tuberculosis in resource poor settings. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tuberculosis (TB) is an important communicable disease affecting the human population world-wide. Despite the efforts of the scientific community, national governments and WHO in controlling the disease, TB still remains a major killer in resource poor settings. New rapid assays and techniques that are simple and cost-effective are urgently needed to identify, treat and understand pathogenesis including the geographical distribution of the disease. The aim of the thesis is to develop a novel genomic mapping tool using Insertion Element, IS6110 that could aid in epidemiological studies of Mycobacterium tuberculosis complex (MTBC) in low and middle income countries. IS6110, a bacterial transposon, plays an essential role in changing the physical and biochemical traits of MTBC. Due to their transposition in TB genomes, they are used as epidemiological markers for differentiation of TB organisms and the mapping of these elements could also shed light on the putative altered function of adjacent genes. In the era of Whole Genome Sequencing (WGS) where repeat elements are difficult to sequence with short read technologies, a rapid and simple method of insertion site mapping using IS6110 FAFLP PCR was developed. This work is aimed at developing a rapid, cost-effective and robust genomic tool box exploiting the IS6110 FAFLP PCR assay that can both identify and characterise the TB genotypes / genetic lineages in any geographical location. For the first time using the assay above, TB samples from Nepal were categorised into different genetic lineages. Fifty-five percent of the samples analysed belong to Principal Genetic Group 1 (PGG1), Beijing and Central Asian strains. Also, new primers were designed targeting the Beijing and the T- groups using the FAFLP derived data that gave rise to the development of rapid lineage specific PCR assays. In addition, it was noticed that 3.9% of the Nepalese strains tested in this research work were likely multi-drug resistant (MDR-TB) using PCR targeting the Rifampicin-resistance-determining region (RRDR) of the rpoB region. It is demonstrated here that IS6110 FAFLP methodology could easily characterise the TB samples into different genetic lineages provided they have more than four IS6110 copies. In addition, lineage specific PCR does not need any expensive instruments or reagents except for PCR blocks and gel visualisers, and could be very effective in the rapid identification of different TB genotypes within hours. These data also add to knowledge about the circulating strains of TB in Nepal, currently a poorly characterised region of the world in this regard, and could help in contact tracing studies by epidemiologists. The IS6110 FAFLP technique thus can be employed in any geographical location to map TB genetic lineages where there is little or no information available on the prevailing TB strains.

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