Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

Abstract

Objectives: The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR-in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results.

Methods: We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer's specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription-polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated.

Results: The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate.

Conclusions: The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

Keywords: COVID-19; Digital droplet polymerase chain reaction; Real-time polymerase chain reaction; SARS-CoV-2.

Figure 1

A, Limit-of-detection study with a 95% (19 of 20) positivity rate at a concentration of 50 copies/mL. Sample 20 failed, with only 1 positive droplet below our cutoff. B, Rate of positivity of internal control (ribonuclease P protein subunit p30 [RPP30]) compared with the number of accepted droplets. The total accepted droplet count is not related to percent positive internal control. Samples with RPP30 < 5% had high-viral-load artifact present.

Figure 2

Examples of severe acute respiratory syndrome coronavirus 2–positive samples. A, All possible clusters displayed and labeled. Each is discrete and easily distinguishable. B, A typical plot of a positive and C, smear artifact observed in cases with high viral load. D, Artifact caused by inhibitors in a positive sample.

Figure 3

A, B, and D compare the concentration of positive droplets vs the cycle threshold (Ct) values for corresponding polymerase chain reaction results. A, Bias toward N1 droplets called positive and N2 called negative at lower Ct values. B, Saturation effect observed at Ct values < 10. C, Comparison of N1-positive droplets with N2-positive droplets in every sample (blue circles) (slope = 0.15; R2 = 0.088). Bias toward N1 or N2 is eliminated when smear artifact data are excluded (red squares) (slope = 0.94; R2 = 0.992). D, Linear relationship for N1 (slope = –0.2475; R² = 0.81) and N2 (slope = –0.2244; R² = 0.81) compared with Ct values > 10.