Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

The protocol is intended to serve as a blueprint for universities and other organizations considering large-scale testing for SARS-CoV-2 or developing preparedness plans for future viral outbreaks.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has caused the deaths of over 6.2 million people to date, with numbers rising every day¹. The gold standard of testing for SARS-CoV-2 is quantitative real-time (RT-q) PCR, with primers designed to target the viral genome, such as nucleocapsid, envelope, spike, and RNA-dependent RNA polymerase genes². At the beginning of the pandemic, sufficient capacity for SARS-CoV-2 testing was severely lacking. It arose from a lack of validated assays, testing components, clinical personnel, and an infrastructure unprepared to rapidly expand to accommodate pandemic-level, mass tes....

Studies performed to optimize the methods for the Early Detection Program were approved by the Michigan State University Institutional Review Board. All figures were reproduced with contrived samples and are representative of the observed human results. No data, information, or results shown in the manuscript are from any participant in the Michigan State University Early Detection Program.

1. Kit production

NOTE: During kit assembly, wear masks, glov.......

The vast majority of samples received by the lab to date have been accepted and passed the initial visual quality control step. The need to reject a sample is limited to reasons that can negatively influence sample processing and/or the overall results for the sample. Specifically, incorrect volume in the tube, consistency, or color not natural to saliva, an absence of ceramic beads used to aid in sample homogenization, and missing barcodes are all reasons to reject a sample (Figure 1).

During sample processing, there are steps requiring careful attention. The initial quality control step which looks at the sample volume, consistency, color, and presence of added beads is critical to the overall success of the process. Tubes with samples that do not contain the correct amount of saliva could produce a false negative, as too little saliva would result in not enough genetic material; conversely, too much saliva would not be in the correct ratio with the RNA buffer and RNA degradation could occur. In rare .......

The authors would like to acknowledge participants in Michigan State University Institutional Review Board approved studies used to optimize the methods (STUDY00004265, STUDY00004383, STUDY00005109), as well as those that went out to collect samples used to test the methods (Dr. Katie Miller, Anna Stoll, Brian Daley, Dr. Claudia Finkelstein). This endeavor was supported by Michigan State University.