Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer

Abstract

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.

Keywords: Aptamer; COVID-19; Glucometer; Point-of-care; Population screening; SARS-CoV-2.

Fig. 1

Overview of proposed point-of-care, aptamer-based COVID-19 assay. SARS-CoV-2 N or S protein-specific biotinylated aptamer is conjugated to a streptavidin-coated magnetic bead (MB) and pre-hybridized with a complementary antisense oligonucleotide strand that is covalently attached to an invertase enzyme. The saliva sample is added to this cocktail (Step 1). Upon binding to the viral antigen or the SARS-CoV-2 virion, the invertase-antisense oligo is released (Step 2). A magnet is used to remove the MB conjugated to the aptamer-antigen complex and the remaining aptamer-antisense-invertase complex (Step 3). The solution containing the released antisense-invertase is then collected and incubated with sucrose (Step 4). Invertase converts sucrose to glucose that is directly readout using a glucometer. The glucose concentration is correlated with the SARS-CoV-2 N or S protein concentration. (Glucometer image adopted with permission from Hangzhou Sejoy Electronics & Instruments ltd, China.)

Fig. 2

Calibration curves for SARS-CoV-2 N and S protein in buffer and saliva measured with a glucometer. Linear calibration curve with subtracted background signal of protein N spiked into (A) buffer and (C) saliva and protein S spiked in (B) buffer and (D) saliva. All measurements taken with n = 3 and error bars represent ±1σ.

Fig. 3

Cross-reactivity study. Assays using analogous proteins with (A) N aptamer complex and (B) S aptamer complex. All proteins were spiked into DPBS at 500 pM. All measurements taken with n = 3 and error bars represent ±1σ.

Fig. 4

Detection of N and S protein using authentic SARS-CoV-2. (A) Schematic of authentic SARS-CoV-2 virus preparation and quantification of viral RNA by ddPCR and infectious units by plaque assay. (B) N and S aptamer/antisense MB complex detection of the SARS-CoV-2 native protein in 1:10 diluted virus culture media. Background media (control) values were subtracted from the measurement results. All measurements taken with n = 3 and error bars represent ±1σ.

Fig. 5

Clinical performance of saliva samples from COVID-19 patients (n=16) and healthy volunteers (n=8). (A) Bar plot of all subjects using S aptamer magnetic bead complex and (B) box and whisker plot of all data points. With a cut-off threshold of 52 mg/dL, this assay has 100% PPA and 100% NPA with RT-qPCR data.