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The development of biosurveillance programs with strong analytical performance and economically accessible protocols is essential for monitoring viral pathogens. Throughout the COVID-19 pandemic, whole-genome sequencing (WGS) has been the prevailing technology for SARS-CoV-2 variant of concern (VOC) detection. While WGS offers benefits, it is a lengthy process, financially and technically straining for scalable viral tracking. The aim of this study was to compare the analytical performance and economic feasibility of WGS and PCR mutation panels for distinguishing six known VOCs: Alpha (B.1.1.7 and Q.4), Gamma (P.1), Delta (B.1.617.2 and AY.4.2), and Omicron. (B.1.1.529.1). In all, 78 SARS-CoV-2-positive samples were collected from April to December 2021 at Northeastern University (Cabot Testing Site, Boston, MA, USA) for genotyping PCR and WGS analysis. MagMax Viral/Pathogen II Nucleic Acid Isolation and TaqPath COVID-19 Combo Kits were used for RNA extraction and SARS-CoV-2 confirmation. VOC discrimination was assessed using two TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assay layouts, and Ion Torrent WGS. In November 2021, the mutation panel demonstrated marked versatility by detecting the emerging Omicron variant reported by South Africa. SNP panel analysis yielded the following 78 VOC identifications: Alpha B.1.1.7 ( N = 20), Alpha Q.4 ( N = 3), Gamma P.1 ( N = 1), Delta B.1.617.2 ( N = 30), Delta AY.4.2 ( N = 3), and Omicron B.1.1.529.1 ( N = 20) with one undetermined ( N = 1) sample. Genotyping mutation panels designated lineages in 77 of 78 samples, 46/78 were confirmed by WGS, while 32 samples failed WGS lineage assignment. RT-PCR genotyping panels offer pronounced throughput and sensitivity and provide an economically advantageous technique for SARS-CoV-2 biosurveillance.IMPORTANCEThe results presented in our manuscript demonstrate how the value of simplistic and reliable molecular assays coupled with the core scientific principle of standardization can be overlooked by the charm of more sophisticated assays and instrumentation. This effect can often be amplified during tumultuous public health events, such as the COVID-19 pandemic. By adapting standardized PCR mutation panels to detect prominently circulating SARS-CoV-2 variants, we were able to better assess the potential health impacts of rising positivity rates and transmission clusters within the Northeastern University population. While several literature publications utilizing genotyping PCR and NGS have a similar scope to ours, many investigations lack sufficiently standardized genotyping PCR and NGS bioinformatics inclusionary/exclusionary criteria for SARS-CoV-2 variant identification. Finally, the economic benefits of standardized PCR mutation panels would allow for global implementation of biosurveillance, rather than reserving biosurveillance to more economically developed nations., Competing Interests: The authors declare no conflict of interest.

During the past two decades, the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER)-related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8-oxoguanine. However, for many 'real-life' studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate 'tissues' such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimisation step in preventing interference by non-specific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5 mg/ml for mouse liver extracts and 1 mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5 μM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

A high drop-out rate prior to the running of a Marathon reduces its value as a spectacle and needlessly causes rejection and antagonism amongst potential runners. This research investigates reasons for pre-race drop-out in the 1983 Glasgow Marathon, and finds that a major cause was injury or illness during training, with a motivational difference existing between runners and non-runners.