Ultrafast PCR Detection of COVID-19 by Using a Microfluidic Chip-Based System

Abstract

With the evolution of the pandemic caused by the Coronavirus disease of 2019 (COVID-19), reverse transcriptase-polymerase chain reactions (RT-PCR) have invariably been a golden standard in clinical diagnosis. Nevertheless, the traditional polymerase chain reaction (PCR) is not feasible for field application due to its drawbacks, such as time-consuming and laboratory-based dependence. To overcome these challenges, a microchip-based ultrafast PCR system called SWM-02 was proposed to make PCR assay in a rapid, portable, and low-cost strategy. This novel platform can perform 6-sample detection per run using multiple fluorescent channels and complete an ultrafast COVID-19 RT-PCR test within 40 min. Here, we evaluated the performance of the microdevice using the gradient-diluted COVID-19 reference samples and commercial PCR kit and determined its limit-of-detection (LoD) as 500 copies/mL, whose variation coefficients for the nucleocapsid (N) gene and open reading frame 1 ab region (ORF1ab) gene are 1.427% and 0.7872%, respectively. The system also revealed an excellent linear correlation between cycle threshold (Ct) values and dilution factors (R2 > 0.99). Additionally, we successfully detected the target RNAs and internal gene in the clinical samples by fast PCR, which shows strong consistency with conventional PCR protocol. Hence, with compact dimension, user-friendly design, and fast processing time, SWM-02 has the capability of offering timely and sensitive on-site molecular diagnosis for prevention and control of pathogen transmission.

Keywords: COVID-19; microchip-based system; microfluidics; point-of-care test; polymerase chain reaction.

Figure 1

(A) Design and configuration diagram of the multiple fluorescent collection module. (B) Outlook and dimension of Real-time PCR Nucleic Acid Detection System SWM-02 and microchip BS-C3-12.

Figure 2

A general control scheme of the SWM-02 microfluidic PCR control system.

Figure 3

Thermal performance of the microheater. (A) Temperature profile in thermal cycling. (B) The linear relationship between electrical resistance and real-time temperature (R² = 0.999) and patterned microheater with heating and sensor terminal.

Figure 4

Fluorescent photos of two 3-well microchips (1000 copies/mL (CPS)) captured by CMOS camera (FAM) at the endpoint of the 1st cycle (A) and the 32nd cycle (B). (C)Average Ct values of N gene, ORF1ab gene, and RNase P. Added concentration of N gene and ORF1ab gene are 10,000, 1000, 500, and 200 CPS per mL. Correspondingly, the dilution factors of reference gene (RNase P) are 20, 200, 400, and 1000. Error bars represent the standard deviation of the 3 replicates.

Figure 5

Average Ct values of the 500 CPS standard samples at six replicates using the ultrafast PCR platform. Error bars represent the standard deviation of the 6 replicates.

Figure 6

After a standard 42-cycle (including 10 pre-cycles without fluorescence collection) rapid RT-PCR, the fluorescence curves and the linear relationship between Ct value and logarithmic sample concentration (Log10) of N gene (A) and ORF1ab gene (B). The standard samples of SARS-CoV-2 are derived from 10,000, 1000, 500, and 200 copies/mL and perform 3 replicates for each concentration.

Figure 7

Ct values of 1~115 clinical samples using SWM-02 ultrafast PCR platform (N gene, ORF1ab gene, and RNase P).