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. 2021 Dec 15;16(12):e0260487.
doi: 10.1371/journal.pone.0260487. eCollection 2021.

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

Justin S Lee  1 , Jason M Goldstein  1 , Jonathan L Moon  1 , Owen Herzegh  1 , Dennis A Bagarozzi Jr  1 , M Steven Oberste  2 , Heather Hughes  1 , Kanwar Bedi  1 , Dorothie Gerard  1 , Brenique Cameron  1 , Christopher Benton  1 , Asiya Chida  1 , Ausaf Ahmad  1 , David J Petway Jr  1 , Xiaoling Tang  1 , Nicky Sulaiman  1 , Dawit Teklu  1 , Dhwani Batra  1 , Dakota Howard  1 , Mili Sheth  1 , Wendi Kuhnert  3 , Stephanie R Bialek  2 , Christina L Hutson  4 , Jan Pohl  1 , Darin S Carroll  1
Affiliations

Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

Justin S Lee et al. PLoS One. .

Abstract

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the "bulk" manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3' end of the N3 probe and the 3' end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the "bulk" material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Next generation sequence data from products of the EUA Kit N1.
34% of reads generated from the EUA-kit N1 NTC reaction with false-positive reactivity mapped to the SARS-CoV-2 Wuhan-Hu-1 reference sequence (highlighted sequence at top) but contained four distinguishing SNPs (highlighted in the mapped reads) consistent with a contaminating template synthesized at CDC around the same time as the kit production (S1a Fig). The N1 primer and probe binding sites are annotated above the reference sequence.
Fig 2
Fig 2
a. The NGS read length distribution (insert sizes after adapter and quality trimming) from the EUA-kit N3 false positive product demonstrating three prominent populations of molecules at 36 bp, 46 bp, and 58 bp in length. All three populations were identified as originating from the N3 assay components and comprise oligonucleotide duplex or triplex molecules (Fig 2b). b. Representative NGS reads from the three most common populations of the EUA-kit N3 false-positive RT-PCR reaction products (Fig 2a). N3 primers and probes are mapped below each read (reverse primer shown in reverse-complement). The 58 bp product and 36 bp products involve the probe and therefore would have generated fluorescence in the absence of template during the reaction. These same populations of products were generated from all three sets of primers and probes (pre-EUA, EUA-kit, commercial vendor).
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Grants and funding

The author(s) received no specific funding for this work.