Specimen Collection and Processing
Beginning in the fall of 2020, all employees and students at the Rockefeller University campus (approximately 1400 persons) were tested at least weekly with a saliva-based PCR test developed in the Darnell Clinical Laboratory Improvement Amendments–Clinical Laboratory Evaluation Program laboratory (approval number, PFI-9216) and approved for clinical use by a New York State emergency use authorization. Protocols for the collection of saliva samples for clinical SARS-CoV-2 testing were reviewed by the institutional review board at Rockefeller University and were deemed not to be research involving human subjects. Institutional review board–approved written informed consent for the analysis of antibody titers was obtained from Patient 1, and the study was conducted in accordance with International Council for Harmonisation Good Clinical Practice guidelines.
In accordance with New York State regulations regarding eligibility, 417 employees who had received a second dose of either the BNT162b2 (Pfizer–BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously were tested between January 21 and March 17, 2021, and weekly testing continued thereafter. The demographic characteristics of these 417 persons and of 1491 unvaccinated persons tested in parallel at Rockefeller University during the same period are shown in Table S1 of the Supplementary Appendix, available with the full text of this article at NEJM.org.
The employees and students were instructed to provide a saliva sample in a medicine cup and transfer 300 μl into a vial containing 300 μl of Darnell Rockefeller University Laboratory (DRUL) buffer (5 M of guanidine thiocyanate, 0.5% sarkosyl, and 300 mM of sodium acetate [pH 5.5]).2 Samples were processed on the Thermo KingFisher Apex system for rapid RNA purification, and complementary DNA (cDNA) was amplified with the use of TaqPath 1-Step RT-qPCR (reverse-transcriptase quantitative PCR) Master Mix (Thermo Fisher Scientific) and multiplexed primers and probes that were validated under a Food and Drug Administration emergency use authorization (Table S2) with the 7500 Fast Dx Real-Time PCR detection system (Applied Biosystems). Samples were considered to be interpretable if the housekeeping control (RNase P) cycle threshold (Ct) was less than 40, and viral RNA was considered to be detected with both viral primers and probes (N1 and N2, detecting two regions of the nucleocapsid [N] gene of SARS-CoV-2) at a Ct of less than 40.
Viral Load Calculation
We calculated the viral load per milliliter of saliva using chemically inactivated SARS-CoV-2 (ZeptoMetrix) spiked into saliva at various dilutions. Extractions and RT-PCR were performed as described previously to determine the corresponding Ct values for each dilution (Fig. S1).
Reverse transcription of RNA samples was performed with the iScript mix (Bio-Rad) according to the manufacturer’s instructions. PCR amplification of cDNA was performed with the use of two primer sets (primer set 1: forward primer 1 [CCAGATGATTTTACAGGCTGC] and reverse primer 1 [CTACTGATGTCTTGGTCATAGAC]; primer set 2: forward primer 2…
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