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

Ham, Rachel E.; Smothers, Austin R; Che, Rui; Sell, Keegan J; Peng, Congyue Annie; Dean, Delphine

doi:10.1128/spectrum.00797-22

Cited by 5 publications

(6 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“… 25 Details of the methods are available elsewhere. 24 − 28 Briefly, saliva is heat treated to 95 °C for 30 min. Then, 2 μL of sample are loaded using open-source sample handlers (Opentrons OT-2) into prepared plates with enzyme mix (New England Biolabs M3006B, M3002B), primers, and probes.…”

Section: Methodsmentioning

confidence: 99%

“…The TigerSaliva multiplex RT-qPCR test targets the N gene of SARS-CoV2 for diagnosis; it is a version of the EUA-approved SalivaDirect protocol . Details of the methods are available elsewhere. − Briefly, saliva is heat treated to 95 °C for 30 min. Then, 2 μL of sample are loaded using open-source sample handlers (Opentrons OT-2) into prepared plates with enzyme mix (New England Biolabs M3006B, M3002B), primers, and probes.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Predicting COVID-19 Infected Individuals in a Defined Population from Wastewater RNA Data

et al. 2022

Self Cite

View full text Add to dashboard Cite

Wastewater surveillance of SARS-CoV-2 RNA has become an important tool for tracking the presence of the virus and serving as an early indicator for the onset of rapid transmission. Nevertheless, wastewater data are still not commonly used to predict the number of infected individuals in a sewershed. The main objective of this study was to calibrate a susceptible-exposed-infectious-recovered (SEIR) model using RNA copy rates in sewage (i.e., gene copies per liter times flow rate) and the number of SARS-CoV-2 saliva-test-positive infected individuals in a university student population that was subject to repeated weekly testing during the Spring 2021 semester. A strong correlation was observed between the RNA copy rates and the number of infected individuals. The parameter in the SEIR model that had the largest impact on calibration was the maximum shedding rate, resulting in a mean value of 7.72 log 10 genome copies per gram of feces. Regressing the saliva-test-positive infected individuals on predictions from the SEIR model based on the RNA copy rates yielded a slope of 0.87 (SE = 0.11), which is statistically consistent with a 1:1 relationship between the two. These findings demonstrate that wastewater surveillance of SARS-CoV-2 can be used to estimate the number of infected individuals in a sewershed.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Predicting COVID-19 Infected Individuals in a Defined Population from Wastewater RNA Data

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although the NGS is the gold standard for differentiating SARS-CoV-2 variants, due to its long turnaround time and high cost per sample, its use is less feasible by diagnostic laboratories [ 26 ]. Therefore, RT-qPCR assays detecting VOCs-specific mutations appear to be a better option for timely clinical applications and population surveillance [ 26 , 38 , 39 , 40 , 41 ]. The present study applied just this method to test a set of more than 1000 samples for SARS-CoV-2 variants.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of SARS-CoV-2 Variants Using RT-qPCRs by Targeting Recurrent Mutation Sites: A Diagnostic Laboratory Experience from Multi-Center Regional Study, August 2020–December 2021, Poland

Wegrzynska

Komiażyk

Walory

et al. 2022

IJMS

View full text Add to dashboard Cite

Rapid identification of SARS-CoV-2 variants is essential for epidemiological surveillance. RT-qPCR-based variant differentiation tests can be used to quickly screen large sets of samples for relevant variants of concern/interest; this study was conducted on specimens collected at 11 centers located in Poland during routine SARS-CoV-2 diagnostics between August 2020 and December 2021. A total of 1096 samples (with CT < 30) were screened for Alpha, Beta, Delta, Kappa and Omicron variants using commercial assays targeting repeat mutation sites. Variants were assigned to 434 (39.6%) specimens; the remaining 662 (60.4%) samples were not classified (no tested mutations detected). Alpha (n = 289; 66.59%), Delta (n = 115; 26.5%), Kappa (n = 30; 6.91%) and Omicron (n = 2; 0.46%) variants were identified and their distribution changed over time. The first Alpha variant appeared in October 2020, and it began to gradually increase its proportion of the virus population by June 2021. In July 2021, it was replaced by the Delta variant, which already dominated by the end of the year. The first Kappa was detected in October 2021, while Omicron was found in December 2021. The screening of samples allowed the determination of epidemiological trends over a time interval reflecting the national COVID-19 waves.

show abstract

“…Samples were stripped of personal identifiers, and one aliquot was used to quantify the viral load of SARS-CoV-2 at the Clemson University Research and Education in Disease Diagnosis and Intervention (REDDI) Lab (CLIA# 42D2193465). The TigerSaliva multiplex RT-qPCR saliva diagnosis test was used to quantify the concentration of the SARS-CoV-2 virus in the clinical samples using previously established methods. − Briefly, the saliva samples were heat-treated at 95 °C for 30 min, and 2 μL was transferred automatically to a 384-well plate by an open-source sample handler (Opentrons OT-2). The plates were loaded into a thermocycler (Bio-Rad CFX 384) for amplification.…”

Section: Methodsmentioning

confidence: 99%

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

Szydlowska,

Pola,

Cai

et al. 2024

ACS Appl. Mater. Interfaces

Self Cite

View full text Add to dashboard Cite

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 highperformance biosensing applications. Here, we present screenprinted 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 10 3 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.

show abstract

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

Cited by 5 publications

References 40 publications

Predicting COVID-19 Infected Individuals in a Defined Population from Wastewater RNA Data

Predicting COVID-19 Infected Individuals in a Defined Population from Wastewater RNA Data

Differentiation of SARS-CoV-2 Variants Using RT-qPCRs by Targeting Recurrent Mutation Sites: A Diagnostic Laboratory Experience from Multi-Center Regional Study, August 2020–December 2021, Poland

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

Contact Info

Product

Resources

About