“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…
Results
Analysis of N1 primers and probes
... Real-time RT-PCR assays were performed on the pre-EUA, EUA-kit, of N1 diagnostic panel oligonucleotides. False reactivity of the N1 oligonucleotides in NTC wells was observed only with the EUA-kit components, but not with the pre-EUA primers and probes...
Based on these findings, and given that the false reactivity of the N1 primers and probes was not seen in subsequent lots of EUA oligonucleotides produced at the CDC, we conclude that the source of N1 false-positive reactivity in the first lot of the EUA diagnostic panel released to public health laboratories was due to contamination of the kits by a synthetic oligonucleotide...
Analysis of N3 primers and probes
All three sets of N3 oligonucleotides tested (pre-EUA, EUA-kit, and commercial vendor) produced some false-positive reactivity of the N3 primers and probes in the NTC wells from RT-PCR (Table 1). The proportion of NTC reactions with false reactivity was highest in the EUA-kit materials...
Without any evidence of contamination, and with consistent amplification of N3 primer-probe complexes across three different sources of oligonucleotides, we conclude that the false-positive reactivity among the N3 components of the diagnostic assay was due to an assay design flaw. Complementarity between the N3 probe and N3 reverse primer resulted in amplification of duplex and triplex molecules, emitting fluorescence during the RT-PCR assay in the absence of target or other template molecules. Given that the rate of false reactivity varied from low (0.5–2%) to high (100%) depending on the source of the oligonucleotides, the frequency of the false reactivity appears to be highly variable and at this time we do not know what factors may influence the outcome.
Discussion: The N1 and N3 components of the first distribution (Lot #20–0121) of the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel suffered from sporadic false-positive reactivity.”
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