Saliva-STAT: Sample-to-answer saliva test for COVID-19

Abstract

Highly accessible and highly accurate diagnostics are necessary to combat rapidly-spreading infectious diseases, such as the recent COVID-19 pandemic. While lateral flow antigen tests have become pervasive, they are insufficiently sensitive to detect early or asymptomatic disease. Nucleic acid amplification tests provide the needed sensitivity, but accessibility of these tests continues to be a challenge due to the need for precise sample processing steps. Here we report a sample-to-answer test for saliva samples (saliva-STAT) that utilizes a battery-powered handheld instrument and a low-cost easily-manufacturable sample cassette to perform a nucleic acid amplification test for viral pathogens. To enable a completely automated assay, we leverage thermally responsive alkane partitions (TRAPs) and paramagnetic beads for virus purification and concentration, as well as reagent addition and mixing. Notably, the saliva STAT easily accommodates directly-dispensed saliva samples (in contrast to microfluidic devices), which is necessary for self-testing. Using the saliva-STAT platform, we demonstrate detection of down to 0.2 copies/μL of SARS-CoV-2 virus in saliva samples. We envision that the saliva-STAT could be used in walk-in clinics, mobile clinics, public testing locations, and in the home. With minor adjustments to the assay, the saliva-STAT platform can easily be adapted for other respiratory viruses, such as Influenza.

Keywords: COVID-19; LAMP; SARS-CoV-2; point-of-care; saliva; sample-to-answer.

Figure 1.

Schematic of the saliva-STAT diagnostic test for the SARS-CoV-2 virus. After a saliva sample is deposited into the funnel attached to a cassette, magnetic microbeads functionalized with aptamers capture virus particles present in the sample. The temperature is increased to melt the eicosane layer, allowing a magnet to pull beads through, isolating virus particles from the saliva sample. Once through, the virus particles are lysed. After lysis, the hexacosane layer is melted, initiating the LAMP reaction.

Fig. 2.

(A) Rendering of the assembled saliva-STAT handheld instrument and exploded view. (B) Photo of the instrument with a sample cassette.

Figure 3.

Images of a cassette with a funnel attachment as paramagnetic beads are pulled downward in a saliva sample. The data represents the bead transfer from the saliva sample to the saliva-TRAP interface. The red color value was extracted from a small window near the top of the cassette.

Figure 4.

(A) Fluorescence readout of the bottom region in a cassette during a hexacosane melt, which causes intercalating fluorescent dye to mix with a solution of DNA. (B) The temperature profile shows that the hexacosane breaches within one minute of when the cassette reaches the melting point (57 °C).

Fig. 5.

LAMP in the saliva-STAT. (A) Representative amplification curves for 500 copies and 0 copies of viral RNA in the cassette under a layer of eicosane, heated and measured in the saliva-STAT instrument. (B) Mean times-to-positive for 0, 5, 50, and 500 copies of RNA in the saliva-STAT cassette under a layer of eicosane, heated and measured in the saliva-STAT instrument (N=3). Error bars represent +/− one standard deviation.

Figure 6.

Compiled saliva-STAT test results using 500 μL saliva samples with varying concentrations of SARS-CoV-2.