Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.

Fig 1. Equipment and initiators for SARS-CoV-2 detection using a miniPCR.

A) The miniPCR® thermocycler. (B) The blueGel® electrophoresis chamber: blueGel® allows visualization of a 15 ml agarose gel using an integrated blue LED array. (C) Agarose gel electrophoresis of the SARS-CoV-2 amplification products. (D) Commercial 96-well plate with COVID-19 controls (artificial DNA samples). (E) Temperature cycling scheme used in our PCR protocol. (F) Three different sets of primers were used to target a gene sequence encoding the SARS-CoV-2 N protein.

Fig 2. Evaluation of the sensitivity of the combined use of a miniPCR® thermal cycler (for amplification) and a plate reader (for determination of the amplification extent).

(A-C) Sensitivity trials using different concentrations of the template (positive control) and three different primers sets (A) N1, indicated in blue; (B) N2, indicated in red; and (C) N3, indicated in yellow. Images of agarose gel electrophoresis of the DNA amplification product generated by targeting three different regions of the sequence coding for SARS-CoV-2 N protein. PCR was performed using a miniPCR® thermocycler. Three different primer sets were used (N1, N2, and N3). The initial template amount was gradually increased from left to right: negative control (lane 1), 625 copies (lane 2), 2.5 × 10³ (lane 3), 1.0 × 10⁴ (lane 4), repetition of 1.0 × 10⁴ (lane 5), 4.0 × 10⁴ (lane 6), 2.0 × 10⁵ DNA copies (lane 7), and molecular weight ladder (lane 8). (D-F) Determination of fluorescence, as measured in a commercial plate reader, for different dilutions of SARS-CoV-2 synthetic DNA templates. Results using three different primer sets are shown: (A) N1, indicated in blue; (B) N2, indicated in red; and (C) N3, indicated in yellow. (G) Summary and comparison of fluorescence readings form synthetic samples of SARS-CoV-2 in a wide span of dilutions. Results using three different primer sets are shown: (A) N1, indicated in blue; (B) N2, indicated in red; and (C) N3, indicated in yellow.

Fig 3. Potential use of a plate reader for quantitation of the initial viral load in a sample and the extent of amplification.

(A) Linear relationship between the natural logarithm of the fluorescence reading and the natural logarithm of the viral load. (B) Results of the simulation of real time PCR in a microplate reader.