A rapid and accessible method to profile and identify antibiotic resistance genes in bacteria using multiplex amplicon panels and Oxford Nanopore Technology : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand
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Date
2023
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Massey University
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Abstract
The spread of antibiotic resistance is severely burdening agriculture and healthcare industries. Bacteria have readily gained resistance to nearly all commercially available antibiotics through the use and overuse of antibiotics in both industries. Current methods to detect resistance genes in bacteria are either outdated and slow or too expensive and inaccessible. A method that is rapid and accessible is necessary to help lessen the burden of antibiotic resistance. Our research aimed to develop a method to rapidly detect antibiotic resistance genes in single-strain bacteria and metagenomic samples. To achieve this, we developed a nested PCR protocol that could enrich a panel of target resistance genes and provide each a genomic context through degenerate primers and Nanopore sequencing. In single-strain bacteria, we can enrich all target antibiotic-resistance genes; an average of 38% reads will provide a genomic context to each gene. Using our novel nested-PCR protocol, we can also enrich all target resistance genes up to 1000-fold in faecal samples compared to metagenomic sequencing. With this nested PCR protocol and Oxford Nanopore as a sequencing platform, we can detect target resistance genes in single-strain bacteria and faecal samples in under 6 hours, making this method rapid, inexpensive, and accessible. Further, we can provide a genomic context to each resistance gene, allowing us to determine whether genes are chromosomal or plasmid-bound.
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Figure 1.2 is re-used with the publisher's permission.