Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

Abstract

Introduction: The declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance.

Methods: Using 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples.

Results: The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes.

Discussion: The employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method's robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method's independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

Keywords: COVID-19 pandemic; Delta/Omicron tracking; Pool testing; SARS-CoV-2 wave surveillance; droplet digital PCR; new variant outbreak.

Figure 1

Assay optimization: influence of sample dilution on assay performance. A Delta sample (CT = 27) was combined with nine WT-SARS-CoV-2 samples (CT values ranging from 14 to 20). The combined pool was then analyzed by ddPCR with the L452R probe (Catalogue # CVAAAAD). We examined the distribution of droplet populations across various dilution conditions with water: (A) undiluted, (B) 1/2 dilution, (C) 1/4 dilution, and (D) 1/30 dilution. The diagrams illustrate the variation in droplet distribution under each dilution condition.

Figure 2

Assay optimization: impact of thermal gradient on annealing temperature. A Delta sample (CT = 24) was combined with nine WT-SARS-CoV-2 samples (CT values ranging from 14 to 20). This pooled sample was then analyzed by ddPCR with the L452R probe (CoV_B1429_L452R Biorad^®). Assays were conducted at three different annealing temperatures: 55°C, 58°C, and 60°C. (A) A 2-D plot displaying the distribution of the droplet populations at each of the three annealing temperatures. The smooth line represents 60°C, the dashed line represents 58°C, and the purple dotted line represents 55°C. (B) 1-D plots of the ddPCR reactions at different temperatures, with blue representing positive droplets for the variant, green for the WT, and gray for negative droplets.

Figure 3

Sensitivity and specificity assessment. Receiver operating characteristic (ROC) curves are illustrated for the following probes: (A) L452R, (B) Q954H, (C) P681R, and (D) P681H. These curves were derived from the analysis of pooled samples consisting of one Va-SARS-CoV-2 sample (CT = 27) and nine WT-SARS-CoV-2 samples (CT values ranging from 14 to 25).