SARS-CoV-2 viral variants can rapidly be identified for clinical decision making and population surveillance using a high-throughput digital droplet PCR assay.

Epidemiologic surveillance of circulating SARS-CoV-2 variants is essential to assess impact on clinical outcomes and vaccine efficacy. Whole genome sequencing (WGS), the gold-standard to identify variants, requires significant infrastructure and expertise. We developed a digital droplet polymerase chain reaction (ddPCR) assay that can rapidly identify circulating variants of concern/interest (VOC/VOI) using variant-specific mutation combinations in the Spike gene. To validate the assay, 800 saliva samples known to be SARS-CoV-2 positive by RT-PCR were used. During the study (July 2020-March 2022) the assay was easily adaptable to identify not only existing circulating VAC/VOI, but all new variants as they evolved. The assay can discriminate nine variants (Alpha, Beta, Gamma, Delta, Eta, Epsilon, Lambda, Mu, and Omicron) and sub-lineages (Delta 417N, Omicron BA.1, BA.2). Sequence analyses confirmed variant type for 124/124 samples tested. This ddPCR assay is an inexpensive, sensitive, high-throughput assay that can easily be adapted as new variants are identified.

Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 and Variants Using Digital Droplet Polymerase Chain Reaction at a Large University and Healthcare System in California.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with different infectivity, transmission potential, and morbidity change the characteristics of local epidemics and affect vaccine effectiveness. As part of the University of Southern California COVID-19 Pandemic Research Center's efforts to understand, control, and inform local community on coronavirus disease 2019 (COVID-19), we implemented a SARS-CoV-2 surveillance program among students, employees, and USC Keck Medical Center patients. We present the epidemiology and distribution of SARS-CoV-2 and its variants among the population. Methods: We used digital droplet reverse-transcriptase polymerase chain reaction (PCR) to analyze in real-time remnant SARS-CoV-2 PCR-positive saliva specimens stored at the USC Keck Medicine laboratory between September 2020 and April 2022. Samples were tested for the original strain (A20) and 9 SARS-CoV-2 variants: α(B.1.1.7, Q.1-Q.8), ß(B.1.351, B.1.351.2, B.1.351.3), γ(P.1, P.1.1, P.1.2), δ(B.1.617.2), δ+(or δ417N), ε(B.1.427 and B.1.429), η(B.1.525), λ(C.37) and ο(B.1.1.529, ΒΑ.1, BA.2). We reviewed deidentified health information from positive cases including demographics, history of COVID-19 (eg, symptoms, hospitalizations, and repeat infections), and COVID-19 vaccination status. Results: We reviewed 1169 cases and determined the variant type of 482 specimens: 77 specimens were original strain, 119 "Delta", 165 "Omicron". The original strain was detected during the third and fourth quarters of 2020. The Delta variant appeared during the second quarter of 2021, whereas Omicron appeared in the fourth quarter of 2021. Conclusions: Prospectively tracking SARS-CoV-2 variants in a university population and a hospital system, utilizing a low-cost, high-throughput PCR assay, was feasible. Local variant monitoring remains important to inform prevention and control efforts among university and clinical settings.

High-precision and cost-efficient sequencing for real-time COVID-19 surveillance.

COVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients' Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.