Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the point-of-care (POC) is crucial for reducing the transmission of coronavirus disease 2019 (COVID-19). Adequate access and availability of POC devices for SARS-CoV-2 detection will significantly reduce the number of people who get infected and thus these POC devices are desperately needed. To address this need, we have been developing a valve-enabled lysis, paper-based RNA enrichment, and RNA amplification device (VLEAD) for detecting SARS-CoV-2. VLEAD enables two critical functions required for POC testing: sample preparation and detection.VLEAD consists of three components integrated in one platform: one buffer unit, one mixing unit, and one detection unit.&nbsp; The buffer unit includes 4 wells for a lysis buffer, a binding buffer, and two wash buffers. The sample preparation process starts by addition of a sample directly to the mixing well, wherein lysis buffer is immediately discharged. The binding buffer is then released to bind virus RNA onto the chromatography paper in the detection unit. This is followed by dispensing of two wash buffers to purify RNA isolated on the paper. All of these sequential steps are controlled by manipulation of ball-based valves. These balls are placed in the openings at the bottom of the wells in the buffer unit, blocking the reagents from flowing. When the mixing unit slides under the buffer unit, a pin in the mixing unit will be aligned with one well at a time, lifting up the ball and allowing the reagents to flow down. Since the reagents can be pre-packaged in the device, all operation steps can be carried out at POC.&nbsp;We chose reverse transcription loop-mediated isothermal amplification (RT-LAMP) as the molecular detection method because of its simple thermal management and great sensitivity and specificity. RT-LAMP is carried out in a commercially available smart coffee mug, Ember Travel Mug, at 62.5oC for 25 min, without the need of recharging. We demonstrated our RT-LAMP assay has a limit of detection of 10 genome equivalents of SARS-CoV-2. The RT-LAMP assay also showed great specificity when tested against related viruses. We used influenza A H1N1 pdm2009 (due to the similar clinical manifestations) and another human coronavirus (CoV-OC43) as a sample to study our SARS-CoV-2 assay, which showed no cross-reactivity. Detection of amplified reaction products is achieved by recording the color change in the presence of SYBR Green by either naked eye or a smartphone camera.We have demonstrated the functions of the device by detecting heat-inactivated SARS-CoV-2 obtained from BEI Resources. This rapid and highly sensitive POC platform for SARS-CoV-2 detection has a potential to help reduce COVID-19 transmission.

Valve-Enabled Sample Preparation and Isothermal Amplification for SARS-Cov-2 Detection at the Point-of-Care

Paper Proceedings: https://asmedigitalcollection.asme.org/IMECE/IMECE2021/volume/85543

ASME 2021 International Mechanical Engineering Congress and Exposition (IMECE2021)

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