Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

Abstract

Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.

Keywords: COVID-19; RT-PCR; diagnosis; droplet digital PCR; multiplex assays; sars-CoV-2.

Figure 1.

Simplex assay droplet separation results. A, B, and C are 1D amplitude results of the ORF1ab, N, and RPP30 targets respectively using various sample categories. D is a representative 2D amplitude result. E is a representative histogram result of a simplex assay

Figure 2.

Duplex assay droplet separation results in the 1D and 2D amplitudes. A) SARS-CoV-2 only sample. B) SARS-CoV-2+ IC sample. C) IC only sample

Figure 3.

Droplet separation in a triplex probe mix assay results. A) SARS-CoV-2+ IC sample. B) SARS-CoV-2 only sample. C) IC only sample

Figure 4.

Droplet separation in a quadruplex amplitude-based assay. A) SARS-CoV-2 spiked in a background of pooled human sample. B) SARS-CoV-2 sample. C) Pooled human sample. D) Negative sample

Figure 5.

Temperature gradient analysis of simplex (A), duplex (B), and triplex probe mix (C) assays from 65°C to 55°C. There was a general increase in droplet amplitude with a decrease in annealing temperature. Optimum separation was achieved at an annealing temperature of 56.9°C in all the assays. Well: A-65°C; B-64.3°C; C-63°C; D-61.1°C; E-58.8°C; F-56.9°C; G-55.7°C; H-55°C

Figure 6.

Reproducibility of replicate wells. A) Intra-assay reproducibility of eight replicate wells using different assays. B) General reproducibility after the replicate wells data of each target in all assays are merged together

Figure 7.

The antiviral activity of remdesivir and Code 30 against SARS-CoV-2 in cell culture. A) Quantified log copies/µL of different targets by ddPCR triplex probe mix assay 24 hpi. B) Inhibition efficiency of remdesivir and Code 30 against SARS-CoV-2 growth in Vero E6 cells

Figure 8.

Flowchart for this research showing steps toward developing multiplex ddPCR assays (including higher order multiplex assays) for SARS-CoV-2. A) Sample handling and preparation workflow. B) ddPCR workflow. C) Data analysis and droplet separation