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Enhanced molecular assays using strand-displacing polymerases and loop-mediated amplification (LAMP) with Bioluminescent Assay in Real Time (BART)

Lasota, Tomasz 2017. Enhanced molecular assays using strand-displacing polymerases and loop-mediated amplification (LAMP) with Bioluminescent Assay in Real Time (BART). PhD Thesis, Cardiff University.
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Abstract

Real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) is becoming a widely accepted method for use in the field of molecular diagnostics. This method makes use of a highly robust core enzymology’s that are tolerant to sample derived inhibitors, along with a priming mechanisms that permit impeccable amplification sensitivities and specificities. These are well documented attributes associated with LAMP, but little is known about factors that drive and interfere with the reverse transcription of RT-LAMP assays. This study aims to address a number of factors that affect RNA amplification, including impedance of priming related to template structure, inhibition of polymerase activities by sample derived inhibitors and the general effect of assay chemistry and primer function with respect to reverse transcription. In addition to the chemistry optimisation and choice of polymerase (DNA / RT), the secondary structure innate within RNA, could significantly affect the efficiency of RT. Priming position and design would also need to be seriously considered with respect to the folding nature of these targets. Overtly, RT-LAMP showed an increased sensitivity to inhibition compared to its DNA counterpart. Similar observations of impeded RNA transcription were made during the development of an internal amplification control (IAC), which was designed to determine the exact inhibitory nature of any tested samples, in tandem with the RT-LAMP. This report clearly discloses that RT amplification controls must be synthesised ‘free of contaminating DNA’, to avoid poor characterisation of first strand DNA synthesis. Alternative ‘non-enzymatic methods’ of reporting amplification in real-time were compared to the bioluminescent assay real-time (BART) reporter; a well-established method of nucleic acid detection and quantification developed and patented by Lumora Ltd, Cambridgeshire (Fortes et al., 2013). Despite BARTs track record for detection of LAMP, its 4 indiscriminate reporting of amplification is of little use for duplexed assay characterisation, such as the IAC / RT-LAMP combined assay. Thus, methods of specific sequence detection were designed that could target single stranded elements of amplified products (STEMs and LOOP structures). It was demonstrated that the mechanism for RT-LAMP fluorescent probing ‘presented here’ was unique to this Thesis and does not fall under the guise of Taqman or other molecular beacon detection mechanisms. Together with BART, this new form of probing was successfully deployed to distinguish between true RT-LAMP and IAC afflicted amplifications. The possibility of utilising the LAMP / BART technologies for microRNA (miRNA) detection was also explored. Even though it is well known that miRNAs have crucial roles in responding to and regulating a wide range of biological and cellular processes, no real headway has been made in developing highly sensitive, low resource methods for their detection. Here we develop novel methods of miRNA detection capable of sensing picomolar levels that also make use of the LAMP and BART chemistry.

Item Type: Thesis (PhD)
Date Type: Submission
Status: Unpublished
Schools: Biosciences
Funders: BBSRC
Date of First Compliant Deposit: 1 May 2018
Last Modified: 16 May 2019 01:53
URI: http://orca.cf.ac.uk/id/eprint/111111

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