ABSTRACT
Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays overall exhibit high sensitivity and specificity. This variant panel can be used as a Research Use Only screening tool for triaging SARS-CoV-2 positive samples prior to whole genome sequencing.
ABSTRACT
The objective was to evaluate the determination of biomarkers of air quality during a mass gathering event at a convention center using a novel air sampling device, AirAnswers(R). This sampler has previously only been used in smaller locations. Here it was run at five crowded locations within the exhibit area for the four days duration of a trade show. The AirAnswers(R) device uses electro-kinetic flow to sample air at high rates and capture bio-aerosols on grounded electrodes in assayable form. Cartridges were removed from the devices and immediately conveyed to the Inspirotec facility in North Chicago, where assays were performed. Biomarkers determined were for allergens and molds previously described for this system. Testing for a new marker, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was also included. The method was validated by determination of capture efficiency with reference to an impinger sampler in a Class III controlled environment chamber. Average capture efficiency for triplicate runs was 14%. One SARS-CoV-2 positive sample as found at the registration area, which was physically separate from the main exhibit area. Cat allergen Fel d 1was found in four of the locations, dog allergen Can f 1 at two. The airborne biomarker of mold proliferation, (1[->]3)-{beta}-D-Glucan, was above the assay range in all locations. The widespread presence of this mold marker could be accounted for by signs of water leakage. A generic 18S RNA marker for mold was developed and similarly showed the presence of mold in all locations, as was a genus marker for penicillium. A species marker for Cladosporium cladosporioides was in two locations. Species markers for Eurotium amstelodami and Trichoderma viride were each in a single location. The main findings were of the widespread presence of mold markers, and the sporadic appearance of SARS-CoV-2. Masking was recommended but not enforced.