Singleplex, multiplex and pooled sample real-time RT-PCR assays for detection of SARS-CoV-2 in an occupational medicine setting

Abstract

For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24-48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid 'multiplex-pooled' strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

Figure 1

SARS-CoV-2 RT-PCR assay development. Assay detects 2 sequences (N1 and N2) from SARS-CoV-2 and a human RNA (RP) as an internal extraction control. (A) Rough Limit of detection (LoD) of positive control RNA spiked into pooled negative nasopharyngeal samples at known copy numbers. Black lines represent the mean, N = 3. The red line denotes the Ct cutoff (38) for a positive result. (B) Comprehensive LoD study of positive control RNA spiked into pooled negative nasopharyngeal samples at known copy numbers. These studies defined 31.25 copies/reaction as the LoD. Black lines represent the mean, N = 20. The red line denotes the Ct cutoff (38) for a positive result. (C) Mock clinical samples created by spiking known concentrations of synthetic SARS-CoV-2 RNA into individual negative nasopharyngeal samples. Samples were blinded to assay personnel. N = 10 for 1 × and 2 × LoD. N = 5 for the 4 × and 8 × LoD. The red line denotes the Ct cutoff (38) for a positive result. (D) 30 positive clinical confirmation samples. Samples were blinded to assay personnel and run concurrently with negative samples. The red line denotes the Ct cutoff (38) for a positive result. Samples with no signal detected during the real time RT-PCR are shown at Ct 45 in the graphs.

Figure 2

SARS-CoV-2 RT-PCR Occupational testing using the Sandia National Laboratories SNL-NM 2019 nCoV Real-Time RT PCR Diagnostic Assay (EUA200481). Total occupational samples tested between April 2020 and June 2021 and the percent positive of these samples.

Figure 3

SARS-CoV-2 RT-PCR multiplex assay development. Assay detects 2 sequences (N1 and N2) from SARS-CoV-2 and a human RNA (RP) as an internal extraction control. (A) Mock clinical samples created by spiking SARS-CoV-2 synthetic RNA at the level of 1 × LoD into individual negative nasopharyngeal samples. N = 20 per technical replicate. The black lines denote the means for each target and replicate. The red line denotes the Ct cutoff (38) for a positive result. (B) 5 negative and 5 positive clinical samples were assessed and compared to the singleplex detection. Samples were blinded to assay personnel. The red line denotes the Ct cutoff (38) for a positive result.

Figure 4

5-sample pool SARS-CoV-2 RT-PCR assay development. (A) Analysis of the first 103 positive samples from 2021 binned to visualize N1 and N2 Ct level. (B) Pools of 5 samples containing one positive sample per pool. Positive samples were chosen to represent the occupational sample population (14 low Ct samples) and to examine the effect of 5 sample pools on high Ct samples (11 samples), which could be missed due to the Ct delay caused by pooling.

Figure 5

Effect of prevalence rate on the number of samples that require repeat testing. (A) Visual display of the first predicted plates for 4% and 12% prevalence in the testing population. The 12% prevalence illustrates the increased likelihood of multiple positives being pooled randomly into the same well. (B) 20 plates were predicted for each prevalence level using random number generation to examine the effect of prevalence on the number of samples that required reanalysis. The colors indicate the number of samples that require rerun of samples independently to identify the positive sample based on 5-sample pools.

Figure 6

Proposed efficient hybrid testing design using two teams to perform both multiplex testing and pooled testing simultaneously. Day 1 denotes the first day of any testing run, for example a Monday or a week. To maintain ~ 24 time to results, all higher risk samples such as symptomatic, known prior contact with a positive individual and recent travel would be run using the multiplex testing. Regular screening would be run by a second team using the 5-sample pooling method. Negative samples from the screening population would be reported the same day. Samples from the positive pools would be set aside and run with the Day 2 multiplex samples. In this way, all samples would report out in ~ 48 h and all high-risk samples would report out in ~ 24 h from collection.