Optimization of tumor-treating field therapy for triple-negative breast cancer cells in vitro via frequency modulation.

PURPOSE: Currently, tumor-treating field (TTField) therapy utilizes a single "optimal" frequency of electric fields to achieve maximal cell death in a targeted population of cells. However, because of differences in cell size, shape, and ploidy during mitosis, optimal electric field characteristics for universal maximal cell death may not exist. This study investigated the anti-mitotic effects of modulating electric field frequency as opposed to utilizing uniform electric fields. METHODS: We developed and validated a custom device that delivers a wide variety of electric field and treatment parameters including frequency modulation. We investigated the efficacy of frequency modulating tumor-treating fields on triple-negative breast cancer cells compared to human breast epithelial cells. RESULTS: We show that frequency-modulated (FM) TTFields are as selective at treating triple-negative breast cancer (TNBC) as uniform TTFields while having a greater efficacy for combating TNBC cell growth. TTField treatment at a mean frequency of 150 kHz with a frequency range of ± 10 kHz induced apoptosis in a greater number of TNBC cells after 24 h as compared to unmodulated treatment which led to further decreased cell viability after 48 h. Furthermore, all TNBC cells died after 72 h of FM treatment while cells that received unmodulated treatment were able to recover to cell number equivalent to the control. CONCLUSION: TTFields were highly efficacious against TNBC growth, FM TTFields showed minimal effects on epithelial cells similar to unmodulated treatment.

Development of a Global Design Education Experience in Bioengineering Through International Partnerships.

The field of engineering is increasingly appreciating the value of diversity for innovative design solutions. Successful engineering depends on our ability to explore constrained parameter spaces for finding the best solutions, and more diverse minds and experiences enable us to explore the entire potential solution space more thoroughly, more quickly, and more creatively. With a goal to expand the diversity of experiences and mindsets in our undergraduate bioengineering curricula, Arusha Technical College (ATC) in Arusha, Tanzania and Clemson University (CU) in Clemson, South Carolina, U.S., have partnered together over the past 5 years to provide intercontinental educational opportunities for undergraduate students, graduate assistants, and faculty. In 2018, CU and ATC collaborated on an international design course targeting undergraduate students in biomedical engineering focused on global health solutions for resource poor communities. Undergraduate students from ATC and CU collaborated on design projects through formal videoconferenced group meetings, e-mail, and various social media platforms. The year ended with a joint design symposium in Arusha where the students presented on their work in a public poster forum. This successful ATC-CU Global Health Design Collaboration pilot year provides a solid model upon which to build. Students reported overall positive experiences and plans to continue in their curriculum to graduation, as well as some ATC and CU students changing their career direction to include global health initiatives.

In vitro studies of heparin-coated magnetic nanoparticles for use in the treatment of neointimal hyperplasia.

Restenosis by neointimal hyperplasia is still an ongoing concern in endovascular surgery. Slowing vascular smooth muscle cell (VSMC) proliferation by reversing the phenotype change, would allow vessel healing and re-endotheliazation. To accomplish this, we have developed heparin-coated magnetic nanoparticles for targeted drug therapy of neointimal hyperplasia. Iron oxide nanoparticles were modified with a poly (ethylene oxide) based coating and then further functionalized with heparin. In vitro experiments were conducted to observe changes in phenotype, metabolic activity, and viability of three relevant cell lines including VSMC, endothelial cells and fibroblasts. Inhibition of proliferation of VSMCs was observed with doses as low as 1 µg/mL Fe of heparin loaded nanoparticle where endothelial cells showed an increase in proliferation in response to treatment. Fibroblasts showed relatively low response. Results suggest proliferation suppression of VSMCs due to phenotype coupled with the increase in endothelial cell proliferation at low doses of heparin coated nanoparticles.

Comparative limb bone loading in the humerus and femur of the tiger salamander: testing the 'mixed-chain' hypothesis for skeletal safety factors.

Locomotion imposes some of the highest loads upon the skeleton, and diverse bone designs have evolved to withstand these demands. Excessive loads can fatally injure organisms; however, bones have a margin of extra protection, called a 'safety factor' (SF), to accommodate loads that are higher than normal. The extent to which SFs might vary amongst an animal's limb bones is unclear. If the limbs are likened to a chain composed of bones as 'links', then similar SFs might be expected for all limb bones because failure of the system would be determined by the weakest link, and extra protection in other links could waste energetic resources. However, Alexander proposed that a 'mixed-chain' of SFs might be found amongst bones if: (1) their energetic costs differ, (2) some elements face variable demands, or (3) SFs are generally high. To test whether such conditions contribute to diversity in limb bone SFs, we compared the biomechanical properties and locomotor loading of the humerus and femur in the tiger salamander (Ambystoma tigrinum). Despite high SFs in salamanders and similar sizes of the humerus and femur that would suggest similar energetic costs, the humerus had lower bone stresses, higher mechanical hardness and larger SFs. SFs were greatest in the anatomical regions where yield stresses were highest in the humerus and lowest in the femur. Such intraspecific variation between and within bones may relate to their different biomechanical functions, providing insight into the emergence of novel locomotor capabilities during the invasion of land by tetrapods.

Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves.

Fibrous development of the extracellular matrix (ECM) of cardiac valves is necessary for proper heart function. Pathological remodeling of valve ECM is observed in both pediatric and adult cardiac disorders. It is well established that intracardiac hemodynamics play a significant role in the morphogenesis of cardiovascular tissues. However, the mechanisms that transduce mechanical forces into morphogenetic processes are not well understood. Here, we report the development of a three-dimensional, in vitro culture system that allows for culture of embryonic valve tissue under specific pulsatile flow conditions. This system was used to investigate the role that fluid flow plays in fibrous ECM expression during valve formation and to test the underlying cellular mechanisms that regulate this mechanotransduction. When cultured under pulsatile flow, developing valve tissues upregulated fibrous ECM expression at both the transcript and protein levels in comparison to no-flow controls. Flow-cultured valve tissues also underwent morphological development, as cushions elongated into leaflet-like structures that were absent in no-flow controls. Furthermore, rhoA, a member of the cytoskeletal actin-regulating GTPase family of proteins, was upregulated and activated by flow culture. Inhibition of the downstream rhoA effector kinase, ROCK, blocked flow-driven fibrous ECM accumulation and tissue stiffening, while the addition of lysophosphatidic acid (LPA), a rhoA activator, stimulated fibrous ECM deposition and tissue stiffening. These results support a prominent role for the rhoA pathway in the mechanotransduction of hemodynamic forces during fibrous remodeling of developing valve tissue. Our results also point to a potential link between regulation of the actinomyosin cytoskeleton and fibrous ECM synthesis in cardiovascular tissues.

Molecular adhesion between cartilage extracellular matrix macromolecules.

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 µm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan-collagen adhesion. Increasing both ionic strength and [Ca(2+)] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca(2+)-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan-collagen interactions. Interestingly, we found a significant increase in aggrecan-collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan-collagen adhesion, together with aggrecan-aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

Enzyme-etching technique to fabricate micropatterns of aligned collagen fibrils.

A technique to tailor-make pre-coated, pre-aligned bovine collagen fibrils, derived from neonatal cardiomyocytes, on the surface of a glass slide into a designated pattern is reported. The unwanted collagen-coated area was erased by a collagenase solution and the tailored area was retained by attaching a microfabricated polydimethylsiloxane stamp directly to the collagen-coated surface. Using this technique, collagen patterns with designated orientations and with clear pattern boundaries and defined shapes were fabricated.

Microfluidic Diffusional Sizing (MDS) Measurements of Secretory Neutralizing Antibody Affinity Against SARS-CoV-2.

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies.

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting.

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ∼10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ∼1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency, and capacity for biological characterization relative to established western blots.

Biolayer-Interferometry-Guided Functionalization of Screen-Printed Graphene for Label-Free Electrochemical Virus Detection.

Additive manufacturing holds promise for rapid prototyping and low-cost production of biosensors for diverse pathogens. Among additive manufacturing methods, screen printing is particularly desirable for high-throughput production of sensing platforms. However, this technique needs to be combined with carefully formulated inks, rapid postprocessing, and selective functionalization to meet all requirements for high-performance biosensing applications. Here, we present screen-printed graphene electrodes that are processed with thermal annealing to achieve high surface area and electrical conductivity for sensitive biodetection via electrochemical impedance spectroscopy. As a proof-of-concept, this biosensing platform is utilized for electrochemical detection of SARS-CoV-2. To ensure reliable specificity in the presence of multiple variants, biolayer interferometry (BLI) is used as a label-free and dynamic screening method to identify optimal antibodies for concurrent affinity to the Spike S1 proteins of Delta, Omicron, and Wild Type SARS-CoV-2 variants while maintaining low affinity to competing pathogens such as Influenza H1N1. The BLI-identified antibodies are robustly bound to the graphene electrode surface via oxygen moieties that are introduced during the thermal annealing process. The resulting electrochemical immunosensors achieve superior metrics including rapid detection (55 s readout following 15 min of incubation), low limits of detection (approaching 500 ag/mL for the Omicron variant), and high selectivity toward multiple variants. Importantly, the sensors perform well on clinical saliva samples detecting as few as 103 copies/mL of SARS-CoV-2 Omicron, following CDC protocols. The combination of the screen-printed graphene sensing platform and effective antibody selection using BLI can be generalized to a wide range of point-of-care immunosensors.

SARS-CoV-2 variant introduction following spring break travel and transmission mitigation strategies.

BACKGROUND: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching. METHODS: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples. RESULTS: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread. CONCLUSIONS: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern.

Covid-19 vaccine effectiveness against general SARS-CoV-2 infection from the omicron variant: A retrospective cohort study.

We aim to estimate the effectiveness of 2-dose and 3-dose mRNA vaccination (BNT162b2 and mRNA-1273) against general Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection (asymptomatic or symptomatic) caused by the omicron BA.1 variant. This propensity-score matched retrospective cohort study takes place in a large public university undergoing weekly Coronavirus Disease 2019 (Covid-19) testing in South Carolina, USA. The population consists of 24,145 university students and employees undergoing weekly Covid-19 testing between January 3rd and January 31st, 2022. The analytic sample was constructed via propensity score matching on vaccination status: unvaccinated, completion of 2-dose mRNA series (BNT162b2 or mRNA-1273) within the previous 5 months, and receipt of mRNA booster dose (BNT162b2 or mRNA-1273) within the previous 5 months. The resulting analytic sample consists of 1,944 university students (mean [SD] age, 19.64 [1.42] years, 66.4% female, 81.3% non-Hispanic White) and 658 university employees (mean [SD] age, 43.05 [12.22] years, 64.7% female, 83.3% non-Hispanic White). Booster protection against any SARS-CoV-2 infection was 66.4% among employees (95% CI: 46.1-79.0%; P<.001) and 45.4% among students (95% CI: 30.0-57.4%; P<.001). Compared to the 2-dose mRNA series, estimated increase in protection from the booster dose was 40.8% among employees (P=.024) and 37.7% among students (P=.001). We did not have enough evidence to conclude a statistically significant protective effect of the 2-dose mRNA vaccination series, nor did we have enough evidence to conclude that protection waned in the 5-month period after receipt of the 2nd or 3rd mRNA dose. Furthermore, we did not find evidence that protection varied by manufacturer. We conclude that in adults 18-65 years of age, Covid-19 mRNA booster doses offer moderate protection against general SARS-CoV-2 infection caused by the omicron variant and provide a substantial increase in protection relative to the 2-dose mRNA vaccination series.

Identification and diagnosis of long COVID-19: A scoping review.

Long COVID-19 (LC-19) is a condition that has affected a high percentage of the population that recovered from the initial disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LC-19 diagnosis is currently poorly defined because of its variable, multisystem, episodic symptoms, and lack of uniformity in the critical time points associated with the disease. Considering the number of cases, workers' compromised efficiency or inability to return to their duties can affect organizations and impact economies. LC-19 represents a significant burden on multiple levels and effectively reduces quality of life. These factors necessitate the establishment of firm parameters of diagnoses to provide a foundation for ongoing and future studies of clinical characteristics, epidemiology, risk factors, and therapy. In this scoping review, we conducted a literature search across multiple publication sites to identify papers of interest regarding the diagnosis of LC-19. We identified 225 records of interest and categorized them into seven categories. Based on our findings, there are only 11 original papers that outline the diagnostic process in detail with little overlap. This scoping review highlights the lack of consensus regarding the definition and, thereby, the LC-19 diagnosis processes. Due to no clear directive and considering the many unknowns surrounding the natural history of the disease and further recovery/sequelae from COVID-19, continued discussion and agreement on a definition/diagnosis will help future research and management of these patients.

A capacitive laser-induced graphene based aptasensor for SARS-CoV-2 detection in human saliva.

SARS-CoV-2 virus induced CoVID-19 pandemic has accelerated the development of diagnostic tools. Devices integrated with electrochemical biosensors may be an interesting alternative to respond to the high demand for testing, particularly in contexts where access to standard detection technologies is lacking. Aptamers as recognition elements are useful due to their stability, specificity, and sensitivity to binding target molecules. We have developed a non-invasive electrochemical aptamer-based biosensor targeting SARS-CoV-2 in human saliva. The aptamer is expected to detect the Spike protein of SARS-CoV-2 wildtype and its variants. Laser-induced graphene (LIG) electrodes coated with platinum nanoparticles were biofunctionalized with a biotin-tagged aptamer. Electrochemical Impedance Spectroscopy (EIS) for BA.1 sensing was conducted in sodium chloride/sodium bicarbonate solution supplemented with pooled saliva. To estimate sensing performance, the aptasensor was tested with contrived samples of UV-attenuated virions from 10 to 10,000 copies/ml. Selectivity was assessed by exposing the aptasensor to non-targeted viruses (hCoV-OC43, Influenza A, and RSV-A). EIS data outputs were further used to select a suitable response variable and cutoff frequency. Capacitance increases in response to the gradual loading of the attenuated BA.1. The aptasensor was sensitive and specific for BA.1 at a lower viral load (10-100 copies/ml) and was capable of discriminating between negative and positive contrived samples (with strain specificity against other viruses: OC43, Influenza A, and RSV-A). The aptasensor detected SARS-CoV-2 with an estimated LOD of 1790 copies/ml in contrived samples. In human clinical samples, the aptasensor presents an accuracy of 72%, with 75% of positive percent of agreement and 67% of negative percent of agreement. Our results show that the aptasensor is a promising candidate to detect SARS-CoV-2 during early stages of infection when virion concentrations are low, which may be useful for preventing the asymptomatic spread of CoVID-19.

Efficacy and selectivity of tumor-treating field therapy for triple-negative breast cancer cells via in-house delivery device.

PURPOSE: Triple-negative breast cancer continues to be one of the leading causes of death in women, making up 7% of all cancer deaths. Tumor-treating electric fields are low-energy, low-frequency oscillating electric fields that induce an anti-proliferative effect on mitotic cells in glioblastoma multiforme, non-small cell lung cancer, and ovarian cancer. Little is known about effects of tumor-treating fields on triple-negative breast cancer and known research for tumor-treating fields only utilizes low (< 3 V/cm) electric field intensities. METHODS: We have developed an in-house field delivery device capable of high levels of customization to explore a much wider variety of electric field and treatment parameters. Furthermore, we investigated the selectivity of tumor-treating field treatment between triple-negative breast cancer and human breast epithelial cells. RESULTS: Tumor-treating fields show greatest efficacy against triple-negative breast cancer cell lines between 1 and 3 V/cm electric field intensities while having little effect on epithelial cells. CONCLUSION: These results provide a clear therapeutic window for tumor-treating field delivery to triple-negative breast cancer.

Repurposing a SARS-CoV-2 surveillance program for infectious respiratory diseases in a university setting.

Standard multiplex RT-qPCR diagnostic tests use nasopharyngeal swabs to simultaneously detect a variety of infections, but commercially available kits can be expensive and have limited throughput. Previously, we clinically validated a saliva-based RT-qPCR diagnostic test for SARS-CoV-2 to provide low-cost testing with high throughput and low turnaround time on a university campus. Here, we developed a respiratory diagnostic panel to detect SARS-CoV-2, influenza A and B within a single saliva sample. When compared to clinical results, our assay demonstrated 93.5% accuracy for influenza A samples (43/46 concordant results) with no effect on SARS-CoV-2 accuracy or limit of detection. In addition, our assay can detect simulated coinfections at varying virus concentrations generated from synthetic RNA controls. We also confirmed the stability of influenza A in saliva at room temperature for up to 5 days. The cost of the assay is lower than standard nasopharyngeal swab respiratory panel tests as saliva collection does not require specialized swabs or trained clinical personnel. By repurposing the lab infrastructure developed for the COVID-19 pandemic, our multiplex assay can be used to provide expanded access to respiratory disease diagnostics, especially for community, school, or university testing applications where saliva testing was effectively utilized during the COVID-19 pandemic.

Guide to assembling a successful K99/R00 application.

The National Institutes of Health's (NIH) K99/R00 Pathway to Independence Award offers promising postdoctoral researchers and clinician-scientists an opportunity to receive research support at both the mentored and the independent levels with the goal of facilitating a timely transition to a tenure-track faculty position. This transitional program has been generally successful, with most K99/R00 awardees successfully securing R01-equivalent funding by the end of the R00 period. However, often highly promising proposals fail because of poor grantsmanship. This overview provides guidance from the perspective of long-standing members of the National Heart, Lung, and Blood Institute's Mentored Transition to Independence study section for the purpose of helping mentors and trainees regarding how best to assemble competitive K99/R00 applications.

Virtual article collection on infectious diseases.

Specifically for COVID-19, we have had several recent articles on SARS-CoV-2. Sohail and Nutini reported on models working to predict the incubation period for SARS-CoV-2 and disease progression. Güler et al. wrote a review of the biophysical and biochemical properties of SARS-CoV-2 which highlighted how the virus's molecular structure allows it to interact and infect cells. These structures are also potential targets for diagnostic and treatment strategies. Lalitha Guruprasad's review on how the various human coronavirus spike proteins interact with human cell proteins and carbohydrate receptors provides further insight on coronavirus-cell interactions as well, and reviews successfully repurposed drugs to combat coronavirus-based diseases.

Effectiveness and protection duration of Covid-19 vaccines and previous infection against any SARS-CoV-2 infection in young adults.

Data on effectiveness and protection duration of Covid-19 vaccines and previous infection against general SARS-CoV-2 infection in general populations are limited. Here we evaluate protection from Covid-19 vaccination (primary series) and previous infection in 21,261 university students undergoing repeated surveillance testing between 8/8/2021-12/04/2021, during which B.1.617 (delta) was the dominant SARS-CoV-2 variant. Estimated mRNA-1273, BNT162b2, and AD26.COV2.S effectiveness against any SARS-CoV-2 infection is 75.4% (95% CI: 70.5-79.5), 65.7% (95% CI: 61.1-69.8), and 42.8% (95% CI: 26.1-55.8), respectively. Among previously infected individuals, protection is 72.9% when unvaccinated (95% CI: 66.1-78.4) and increased by 22.1% with full vaccination (95% CI: 15.8-28.7). Statistically significant decline in protection is observed for mRNA-1273 (P < .001), BNT162b2 (P < .001), but not Ad26.CoV2.S (P = 0.40) or previous infection (P = 0.12). mRNA vaccine protection dropped 29.7% (95% CI: 17.9-41.6) six months post- vaccination, from 83.2% to 53.5%. We conclude that the 2-dose mRNA vaccine series initially offers strong protection against general SARS-CoV-2 infection caused by the delta variant in young adults, but protection substantially decreases over time. These findings indicate that vaccinated individuals may still contribute to community spread. While previous SARS-CoV-2 infection consistently provides moderately strong protection against repeat infection from delta, vaccination yields a substantial increase in protection.

Identifying SARS-CoV-2 Variants of Concern through Saliva-Based RT-qPCR by Targeting Recurrent Mutation Sites.

SARS-CoV-2 variants of concern (VOCs) continue to pose a public health threat which necessitates a real-time monitoring strategy to complement whole genome sequencing. Thus, we investigated the efficacy of competitive probe RT-qPCR assays for six mutation sites identified in SARS-CoV-2 VOCs and, after validating the assays with synthetic RNA, performed these assays on positive saliva samples. When compared with whole genome sequence results, the SΔ69-70 and ORF1aΔ3675-3677 assays demonstrated 93.60 and 68.00% accuracy, respectively. The SNP assays (K417T, E484K, E484Q, L452R) demonstrated 99.20, 96.40, 99.60, and 96.80% accuracies, respectively. Lastly, we screened 345 positive saliva samples from 7 to 22 December 2021 using Omicron-specific mutation assays and were able to quickly identify rapid spread of Omicron in Upstate South Carolina. Our workflow demonstrates a novel approach for low-cost, real-time population screening of VOCs. IMPORTANCE SARS-CoV-2 variants of concern and their many sublineages can be characterized by mutations present within their genetic sequences. These mutations can provide selective advantages such as increased transmissibility and antibody evasion, which influences public health recommendations such as mask mandates, quarantine requirements, and treatment regimens. Our RT-qPCR workflow allows for strain identification of SARS-CoV-2 positive saliva samples by targeting common mutation sites shared between variants of concern and detecting single nucleotides present at the targeted location. This differential diagnostic system can quickly and effectively identify a wide array of SARS-CoV-2 strains, which can provide more informed public health surveillance strategies in the future.