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  • Publication
    Nucleic acid extraction technologies and sustainability
    (University College Dublin. School of Chemistry, 2022) ;
    0000-0001-5854-7365
    Infection and disease detection has progressed rapidly with the introduction of sensitive and specific molecular diagnostic assays employing nucleic acid amplification tests(NAATs). This is relevant given the current severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) pandemic and the prevalence of cancers worldwide. Before a diagnosis can be made clinical samples must be obtained, stabilised for transit to the testing site and processed to acquire the viral/tumour nucleic acids(NA) for the purposes of molecular testing. Herein the impact of viral sample storage and more sustainable NA extraction methods on the subsequent molecular tests utilising gold standard NAAT methodology, quantitative reverse transcription-polymerase chain reaction(qRT-PCR), were examined. The qRT-PCR assay firstly converts viral RNA to complementary DNA(cDNA) and secondly exponentially amplifies a target sequence. In this manner, approaching single molecule quantities of RNA target can be detected from patient samples. qRT-PCR is influenced by sample quality with factors such as sample acquisition, storage and cold chain maintenance being of key importance. In the present work, studies have been performed to determine how the sensitivity of SARS-CoV-2 qRT-PCR detection is impacted by storage conditions such as viral transport media(VTM) and repeated sample freeze-thawing. The objective was to improve the capability to reproducibly detect SARS-CoV-2 by identifying issues with testing that could arise from sparce resources and poor cold chain maintenance. VTM selection was shown to influence the sensitivity and reproducibility of synthetic single-stranded RNA(ssRNA) and double-stranded DNA(dsDNA) detection via qRT-PCR. The best results were obtained from ssRNA samples stored in Hanks’ balanced salt solution(HBSS) and phosphate buffer saline-glycerol(PBS-G). Inclusion of epithelial cells in the sample improved qRT-PCR sensitivity. Repeated sample freeze-thawing reduced qRT-PCR sensitivity with two noted exceptions. It was concluded that VTM selection is important for sensitivity of SARS-CoV-2 molecular diagnostic assays as VTM can negatively impact ssRNA genome stability. Poor sample storage conditions similarly negatively influenced ssRNA stability and downstream molecular testing. This research suggests PBS-G be used as a VTM in SARS-CoV-2 testing for high levels of sample stability and sensitivity. The extraction of RNA from samples is a key pre-processing step to increase qRT-PCR sensitivity. This is achieved by lysing all bio-membranes in the sample to release the RNA into bulk solution for further downstream isolation and purification. Membrane lysis is frequently accomplished through the use of lysis buffers(LB). LB are composed of several agents that denature cells; surfactants, chaotropic salts, and agents that inhibit NA degradation. Triton X-100 is a widely used surfactant in LB formulations, however is a member on the European Union REACH list due to associated environmental concerns. To address this, RNA extractions were performed using LBs supplemented with Triton X-100 and other more sustainable chemical alternatives. qRT-PCR analysis was used to compare the levels of cell lysis for each LB. These modifications aimed to increase the sustainability of the RNA extraction process. The inclusion of surfactant in LB formulations, with the exception of 3% Triton X-100, showed no significant(p<0.05) increase in cell lysis, thus no improvement to qRT-PCR results. RNA was extracted from a matrix containing up to 30mg/mL serum albumin to examine the role of the sample matrix. Increased protein concentration negatively impacted qRT-PCR results, however this was associated with increased RNase activity and not the presence of the protein itself. To conclude it is suggested that clinical LB formulations be revised and that serum-containing RNA samples be treated with RNase inhibitors in a timely manner to achieve sensitive qRT-PCR analysis.
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