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

Jing Xu, MD, PhD; Timothy Kirtek, MD; Yan Xu, PhD; Hui Zheng, PhD; Huiyu Yao, PhD; Emily Ostman, MS; Dwight Oliver, MD; James S. Malter, MD; Jeffrey R. Gagan, MD, PhD; Jeffrey A. SoRelle, MD

Disclosures

Am J Clin Pathol. 2021;155(6):815-822. 

In This Article

Abstract and Introduction

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.

Introduction

The global coronavirus disease 2019 (COVID-19)[1] pandemic continues to pose a serious global public health threat. The COVID-19 pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),[2] is a single-stranded RNA Betacoronavirus with a 26-kilobase genome. Molecular detection of SARS-CoV-2 targeting the viral genes (eg, Orf1a/b, E, S, N genes) is currently the gold standard for assessing acute infection.[3–7]

In the United States, the first clinical assay for SARS-CoV-2 was developed by the Centers for Disease Control and Prevention (CDC)[3] and approved under a Food and Drug Administration (FDA) Emergency Use Authorization (EUA). As of the first quarter of 2021, multiple testing platforms have obtained EUA and been clinically implemented to diagnose SARS-CoV-2 infection. Although these point-of-care tests are rapid, many are limited by moderate sensitivity and high false-negative rates.[8] Real-time polymerase chain reaction (RT-PCR) platforms can detect low levels of virus with high throughput, but weak positives (cycle number [CN] > 35) can be difficult to distinguish from technical artifacts after many PCR cycles. Droplet digital PCR (ddPCR) is an orthogonal method designed to detect and measure precise copy numbers of nucleic acid, but it can be applied to detect extremely low levels of nucleic acid, as well. The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first assay in this class to receive EUA (Precigenome is the only other company with a ddPCR EUA-approved assay), but data characterizing its clinical performance are limited. One study observed that ddPCR has a sensitivity of 93.33% and a specificity of 100% for both the N1 and N2 gene regions of SARS-CoV-2.[9] In this study, we report the limit of detection, artifactual findings, and correlation with other clinically validated methods. Overall, ddPCR is a highly sensitive approach capable of resolving borderline results with reasonable throughput and cost. With its sensitivity, it is well suited to pooled testing as well.

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