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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
1 Step MM, no ROX	Thermo Fisher	A28523
1.2 mlDeep well Plates	Fisher	AB0564
100 mL reagent reservoirs	Corning	4872
2.8 mm Ceramic Beads	OMNI	19-646
25 ml conical w/screw cap	VWR	76338-496
50mL V bottom reservoirs	Costar	4870
5430-High-Speed Centrifuge	Eppendorf	22620601
5ml Eppendorf Tube	Fisher	14282300
8 strip tubes for QuantStudio	life technologies	4316567
Beta Mercaptoethanol	Fisher	AC125472500
Ethanol 200 Proof, Molecular Biology Grade	Fisher	BP28184
Microamp Endura Optical 96-well fast clear reaction plate with barcode	life technologies	4483485
Microamp Fast Optical 96 well plate	Fisher	4346906
Mini Microcentrifuge	Corning Medical	6770
optical caps for strip tubes	life technologies	AB-1820
Optical Film	Thermo Fisher	4311971
PCR plate sealing film Non-optical	Fisher	AB-0558
PCR Plate semi-skirted	Fisher	14230244
QuantStudio 3 Real-Time PCR System, 96-well, 0.1 mL	Thermo Fisher	A28136
Quick RNA Viral Kit confirmation	Zymo	R1035
Reagent Reservoir, 100ml	DOT	229298
RNA Shield	Zymo	R1200-1L
Small Biohazard Bags	Fisher	180000
Taqpath RTPCR COVID19 kit	Thermo Fisher	A47814
Thermo Scientific Sorvall ST4R Plus Centrifuge	Thermo Fisher	75009525
Transfer Pipet	Fisher	22170404
Viral 96 Kit	Zymo	R1041
Vortex Mixer	Fisher	2215414