LE3 .A278 2022

2022

Leblanc, Jason
Davidson, Ross
Wilson, Brian

Acadia University

Bachelor of Science

Honours

Biology

Biology

The novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus is the causative agent of the coronavirus disease-19 (COVID-19) pandemic, that has caused economic collapse and millions of deaths world-wide. During the pandemic, molecular surveillance became imperative as it not only provided key information such as, pathogenicity, transmission, circulation and evolution of the virus, but was also used to ensure quality assurance diagnostics. As nucleic acid amplification tests (NAATs) were the primary method for SARS-CoV-2 detection, sequence mismatches between circulating SARS-CoV-2 strains and NAAT target sites can lead to reduced sensitivity and diagnostic failure. Thankfully, using bioinformatics, primer/probe sequences used in NAATs can be compared to published SARS-CoV-2 genomic data to identify any discrepancies. Unfortunately, due to the proprietary nature of commercial tests, this can only be done for laboratory developed tests (LDTs). In order to define areas to monitor in subsequent mutations analyses, this study aimed to define the precise genomic locations of target genes (i.e., E and N2) used in a commonly used commercial NAAT, the Xpert Xpress’ SARS-CoV-2. First, amplicons produced from reverse transcription polymerase chain reactions (RT-PCR) using overlapping primer pairs (i.e., amplicon tiling) spanning E and N2 genes were subjected to Xpert assay, to estimate the target regions. To further refine the minimal area required for Xpert positivity, additional various combinations of overlapping forward and reverse primers (i.e., primer tiling) were used within the area identified by amplicon tiling. Primer yielding positive results were aligned to a reference genome to identify primer locations. Altogether, the target regions for the Xpert E and N2 genes were estimated between 26269 to 26381 and 29164 to 29230, respectively. These regions were confirmed with next-generation sequencing using amplicons retrieved from an Xpert reaction tested ion a positive clinical specimen. Based on our results, amplicon and primer tiling proves to be a sequencing-independent technique to estimate the genomic location of undisclosed commercial assay targets. Paired with a method of mutation assessment, this would provide a framework for the maintenance of quality assurance in clinical diagnostic testing and could be applied to SARS-CoV-2 assays, as well as any molecular method.

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https://scholar.acadiau.ca/islandora/object/theses:3759