DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2

Abstract

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 μl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/μl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Fig 1. Limit of detection (LOD).

Determination of LOD with (A) phenol and (B) column-based extraction methods and confirmation of LOD using (C) phenol and (D) column-based extraction methods in 20 replicates and (E) column-based extraction method in 20 separate extractions from unique saliva samples. Black bars = N1 primer, open bars = N2 primer, gray bars = RNase P primer. Error bars = 1 standard deviation.

Fig 2. Assay performance with two extraction methods.

Specimen matrix spiked with specified concentration of synthetic RNA and extracted using A. phenol and B. column-based methods. Black bars = N1 primer, open bars = N2 primer, gray bars = RNase P primer. Error bars = 1 standard deviation.

Fig 3. Correlation of NP samples on Cepheid Xpert Xpress SARS-Cov-2 platform versus phenol extraction in DRUL buffer.

Fig 4. Stability of viral RNA.

Stability of RNA assessed with (A) primer set 1 and (B) primer set 2 in DRUL buffer. Black bars = overnight incubation, open bars = after 7 days of incubation. C. Stability assessed at 0°C (black bars), 25°C (open bars), and 38°C (gray bars) and saliva alone (striped bars). Error bars = 1 standard deviation.

Fig 5. Inactivation of virus in DRUL buffer.

Huh-7.5 cell lysis assessed at day 3 (A) and day 5 (B) after incubation with DRUL (black bars) or AVL buffer (open bars) and human coronavirus E229 at buffer:virus volume ratios ranging from 1:2 to 1:10. Insert table shows viral concentration at each ratio. C. Huh-7.5 cell lysis assessed at days 3 (black bars) and 5 (open bars) after incubation with human coronavirus E229 exposed to DRUL buffer for 60 minutes, 10 minutes, or 10 seconds.