Manually pressurized droplet digital PCR chip for rapid SARS-CoV-2 diagnostics

Abstract

Droplet digital PCR (ddPCR) is a technique in which PCR reaction is divided into thousands of nanoliter-sized droplets and has proven to be a great tool in virus diagnostics. Compared to the gold standard system quantitative real-time PCR (RT-qPCR), ddPCR functions particularly well when dealing with samples with low template counts, such as viral concentration. This feature makes the technique suitable for early detection of the virus. In this study, a novel portable PDMS ddPCR chip is introduced. The chip functions without external pumps using manual pressurization with a multichannel pipet. The created droplets are monodispersed and form a monolayer on the chip's collection chamber, from where they can be effortlessly imaged. Droplets were analyzed and counted using artificial intelligence. The use of the manually pressurized chip was demonstrated for a SARS-CoV-2 assay, which takes advantage of isothermal strand invasion-based amplification (SIBA) technology, allowing quick and accurate, even point-of-care analysis of the sample. The results demonstrate that SIBA assays can be divided into nanoliter-sized droplets and used as quantitative assays, giving an approximation of the samples' viral count.

FIG. 1.

Droplet production on a manually pressurized chip. (a) Schematic picture of the chip, where the green arrow marks the inlet for the reaction mixture, gray arrows mark the inlet for the oils, yellow arrow marks the outlet, and blue square shows the T-junction, (b) pipet and chip, (c) T-junction and droplet formation, (d) uniform droplets, (e) and (f) full chips right after droplet formation: (e) from meander chip and (f) from diamond chip. Figure created with BioRender.

FIG. 2.

Differing size of the droplets. (a) Diagram of droplet diameters when using an empty chip, (b) diagram of droplet diameters while using prefilled chip, (c) slow push rate droplets, (d) fast flow rate droplets, e.g., a diagram of how different flow rates affected the droplet size: bigger droplets are in the “slow” category, but the overage size does not vary significantly between slow and fast flow rates. Figure created with BioRender.

FIG. 3.

Limit of detection: (a) 1 copy of RNA, (b) 5 copies of RNA, (c) 20 copies of RNA, (d) whole chip image of 50 copies of RNA, (e) whole chip image of 100 copies of RNA. (f) Expected vs counted positive droplets. The graph is based on 17 experiments; two failed experiments are removed from the data shown here. Figure created with BioRender.

FIG. 4.

RT-qPCR runs: (a) Luna qPCR reaction with dilution series of the SARS-CoV-2 RNA template, (b) real-time SIBA reaction with dilution series of RNA template. Negative reactions are not shown here, but all the repetitions were negative. Table III shows the average time to get positive results from each concentration. Concentration duplications are shown in the same color. Figure created with BioRender.

FIG. 5.

Digital droplet SIBA on manually pressurized chip. (a) Whole chip image illustrating positive and negative droplets and some size differences between the droplets, (b) example of a positive reaction, (c) gray-scale distribution of the positive and negative droplets by Aiforia, (d) example of a negative reaction. Figure created with BioRender.