Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers
- BioFrontiers Institute, University of Colorado Boulder, United States
- Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, United States
- Darwin Biosciences Inc, United States
- Department of Mechanical Engineering, University of Colorado Boulder, United States
- Interdisciplinary Quantitative Biology Graduate Program, University of Colorado Boulder, United States
- Department of Biochemistry, University of Colorado Boulder, United States
- Howard Hughes Medical Institute, University of Colorado Boulder, United States
- Department of Integrative Physiology, University of Colorado Boulder, United States
- Wardenburg Health Center, University of Colorado Boulder, United States
Figures
Figure 1 with 2 supplements
Optimized strategy for controlling natural variability in saliva pH.
(A) Here, saliva samples from 96 different individuals are analyzed for the prevalence of natural acidity extreme enough to trigger the pink-to-yellow color change of phenol red even before …
Figure 1—figure supplement 1
Optimized heat inactivation for safely detecting SARS-CoV-2 in human saliva.
(A) This experiment shows that heating at 95°C for 10 min degrades viral RNA when it is not in the form of virions. Saliva samples were diluted 1:1 with saliva stabilization solution. In vitro …
Figure 1—figure supplement 2
Saliva samples are stable at 4°C for at least 4 days before processing, if stored in saliva stabilization solution.
(A) Schematic of the experimental conditions. (B) RT-LAMP reaction result before and after the isothermal amplification. Saliva samples were spiked with heat-inactivated SARS-CoV-2 virions at the …
Figure 2 with 1 supplement
Optimized RT-LAMP primer sets for detecting SARS-CoV-2 in human saliva.
(A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E [Rabe and Cepko, 2020], ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2× saliva stabilization …
Figure 2—figure supplement 1
Saliva TwoStep primers will detect most or all currently circulating viral variants of concern.
(A) Genome map of SARS-CoV-2 with the regions targeted RT-LAMP primers highlighted in red. SARS-CoV-2 genome map is adapted from BioRender. (B) Sequence alignments of regions of the key SARS-CoV-2 …
Figure 3 with 2 supplements
The test limit of detection is 200 virions/μL.
(A) Saliva samples were spiked with the indicated concentrations of heat-inactivated SARS-CoV-2 virions (top) before being diluted 1:1 with 2× saliva stabilization solution. Samples were then heated …
Figure 3—figure supplement 1
Saliva stabilization solution containing NaOH does not lower sensitivity of colorimetric RT-LAMP detection of SARS-COV-2.
(A) Here, the detection limit of the RT-LAMP assay was assessed in the absence of any saliva or saliva stabilization solution . This was explored in order to determine whether there might be …
Figure 3—figure supplement 2
Blinded sample evaluation.
Plain saliva, or saliva spiked with heat-inactivated SARS-CoV-2 virions at different concentrations, was heated at 95ºC for 10 minutes. Samples were then analyzed using RT-LAMP by a researcher that …
Figure 4 with 2 supplements
Evaluation of RT-LAMP on SARS-CoV-2-positive saliva samples from individuals with no reported symptoms at the time of sample collection.
(A) We re-analyzed university saliva samples that had been previously analyzed for SARS-CoV-2 using quantitative RT-PCR with a primer set against the N gene of SARS-CoV-2 (see Materials and methods).…
Figure 4—figure supplement 1
Quantitative RT-PCR standard curve used to determine the Ct value to virion/μL calculation.
10,000 copies/µL of heat deactivated SARS-CoV-2 virus was spiked into negative saliva specimens from six different volunteers and incubated for 30 min at 95°C. Samples were diluted to indicate …
Figure 4—figure supplement 2
Near normal distribution of quantitative RT-PCR raw Ct values of SARS-CoV-2 N gene from positive individuals.
Between September 16–25, 2020, 8836 saliva samples were screened for SARS-CoV-2 using the direct quantitative RT-PCR method. (A) The amplification of SARS-CoV-2 N gene is detected in 347 samples and …
Figure 5
Assessment of Saliva TwoStep against a nasal swab test.
(A) Matched nasal swabs and saliva from 54 individuals were analyzed (all of whom were SARS-CoV-2 positive at the time that these samples were collected, as verified by the saliva quantitative …
Figure 6 with 1 supplement
Two step detection of SARS-CoV-2 in saliva.
(Upper half) Step 1: Prepare saliva. Person provides 1 mL of saliva, and 1 mL of 2× saliva stabilization solution is then added to it. (This sample can be processed immediately or stored in the …
Figure 6—figure supplement 1
Diagrams of the saliva stabilization solution dispensing apparatus.
(A) CAD model of dispensing apparatus showing components. Custom solution dispensing apparatus fabricated from machined and solvent welded 236-in polycarbonate (Tuffak). Polycarbonate is chosen for …
Author response image 1
Tables
Table 1
Summary of RT-LAMP evaluation in human samples.
| RT-LAMP Primers | |||||||
|---|---|---|---|---|---|---|---|
| AS1E | CU-N2 | ||||||
| No. of samples | No. of positives | Agreement | No. of positives | Agreement | |||
| Quantitative RT-PCR (SARS-CoV-2 N) | Negative | 295 | 0 | 295/295 (100%) | 0 | 295/295 (100%) | |
| Positive (levels of viral load: Virions/μL) | 4000 | 82 | 82 | 82/82 (100%) | 82 | 82/82 (100%) | |
| 2000 | 97 | 97 | 97/97 (100%) | 94 | 94/97 (96.9%) | ||
| 1000 | 118 | 117 | 117/118 (99.2%) | 110 | 110/118 (93.2%) | ||
| 800 | 123 | 122 | 122/123 (99.2%) | 112 | 112/123 (91.1%) | ||
| 400 | 143 | 139 | 139/143 (97.2%) | 129 | 145/173 (90.2%) | ||
| 200 | 168 | 158 | 158/168 (94.0%) | 142 | 142/168 (84.5%) | ||
