CRISPR-based and RT-qPCR surveillance of SARS-CoV-2 in asymptomatic individuals uncovers a shift in viral prevalence among a university population (preprint)

Background: The progress of the COVID-19 pandemic profoundly impacts the health of communities around the world, with unique impacts on colleges and universities. Transmission of SARS-CoV-2 by asymptomatic people is thought to be the underlying cause of a large proportion of new infections. However, the local prevalence of asymptomatic and pre-symptomatic carriers of SARS-CoV-2 is influenced by local public health restrictions and the community setting. Objectives: This study has three main objectives. First, we looked to establish the prevalence of asymptomatic SARS-CoV-2 infection on a university campus in California. Second, we sought to assess the changes in viral prevalence associated with the shifting community conditions related to non-pharmaceutical interventions (NPIs). Third, we aimed to compare the performance of CRISPR- and PCR-based assays for large-scale virus surveillance sampling in COVID-19 asymptomatic persons. Methods: We enrolled 1,808 asymptomatic persons for self-collection of oropharyngeal (OP) samples to undergo SARS-CoV-2 testing. We compared viral prevalence in samples obtained in two time periods: May 28th-June 11th; June 23rd-July 2nd. We detected viral genomes in these samples using two assays: CREST, a CRISPR-based method recently developed at UCSB, and the RT-qPCR test recommended by US Centers for Disease Control and Prevention (CDC). Results: Of the 1,808 participants, 1,805 were affiliates of the University of California, Santa Barbara, and 1,306 were students. None of the tests performed on the 732 samples collected between late May to early June were positive. In contrast, tests performed on the 1076 samples collected between late June to early July, revealed nine positive cases. This change in prevalence met statistical significance, p = 0.013. One sample was positive by RT-qPCR at the threshold of detection, but negative by both CREST and CLIA-confirmation testing. With this single exception, there was perfect concordance in both positive and negative results obtained by RT-qPCR and CREST. The estimated prevalence of the virus, calculated using the confirmed cases, was 0.74%. The average age of our sample population was 28.33 (18-75) years, and the average age of the positive cases was 21.7 years (19-30). Conclusions: Our study revealed that there were no COVID-19 cases in our study population in May/June. Using the same methods, we demonstrated a substantial shift in prevalence approximately one month later, which coincided with changes in community restrictions and public interactions. This increase in prevalence, in a young and asymptomatic population which would not have otherwise accessed COVID-19 testing, indicated the leading wave of a local outbreak, and coincided with rising case counts in the surrounding county and the state of California. Our results substantiate that large, population-level asymptomatic screening using self-collection may be a feasible and instructive aspect of the public health approach within large campus communities, and the almost perfect concordance between CRISPR- and PCR-based assays indicate expanded options for surveillance testing

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2 (preprint)

Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material (preprint)

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point- of-care solution to increase the distribution of COVID-19 surveillance.