Digital Microfluidic Multiplex RT-qPCR for SARS-CoV-2 Detection and Variants Discrimination

Abstract

Continuous mutations have occurred in the genome of the SARS-CoV-2 virus since the onset of the COVID-19 pandemic. The increased transmissibility of the mutated viruses has not only imposed medical burdens but also prolonged the duration of the pandemic. A point-of-care (POC) platform that provides multitarget detection will help to track and reduce disease transmissions. Here we detected and discriminated three genotypes of SARS-CoV-2, including the wildtype and two variants of concern (VOCs), the Delta variant and Omicron variant, through reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a digital microfluidics (DMF)-based cartridge. Upon evaluating with the RNA samples of Omicron variant, the DMF RT-qPCR presented a sensitivity of 10 copies/μL and an amplification efficiency of 96.1%, capable for clinical diagnosis. When spiking with SARS-CoV-2 RNA (wildtype, Delta variant, or Omicron variant) and 18S rDNA, the clinical analog samples demonstrated accurate detection and discrimination of different SARS-CoV-2 strains in 49 min.

Keywords: COVID-19; Delta variant; Omicron variant; RT-qPCR; SARS-CoV-2; digital microfluidics; electrowetting.

Figure 1

Digital microfluidic (DMF) cartridge for SARS-CoV-2 variants detection by RT-qPCR. (a) The top PC plate had five inlets for reaction mix solutions and an inlet for oil; the bottom PCB plate contained electrodes. (b) The five droplets generated from the reservoirs (indicated with rightwards arrows) underwent RT at 48 °C and then qPCR by shuttling between the 95 °C and 60 °C temperature zones (indicated with left right arrows) for the detection of SARS-CoV-2 wildtype (droplet A), Delta variant (droplet B), Omicron variant (droplet C), human 18S rRNA (droplet D) genes, and NTC (droplet E).

Figure 2

Four possible valid diagnostic results, wildtype, Delta, and Omicron positive and negative, from the DMF RT-qPCR. (a) Demonstrative amplification curves for different infection cases. (b) Representative end-point fluorescence signals (+/–) and images of the five droplets A–E for different infection cases.

Figure 3

RT-qPCR of serially diluted SARS-CoV-2 Omicron variant RNA on the DMF cartridge. (a) RT-qPCR amplification curves of delta fluorescence intensity (ΔFI) against cycle number for various template concentrations. (b) Standard curve and efficiency on-chip.

Figure 4

Demonstration of detection and discrimination of SARS-CoV-2 wildtype, Delta variant, and Omicron variant on the DMF cartridge. Amplification curves and initial (cycle 0) and end-point (cycle 45) fluorescence images of RT-qPCR for clinical analog samples representing (a) wildtype infection, (b) Delta variant infection, (c) Omicron variant infection, and (d) no infection.