Table 2. Sequencing results of RT-PCR products demonstrated the source of false reactivity in N1 and N3 components.
| RT-PCR Componentsi | Reagent Sourceii | % Reads Mapped to Referenceiii | % Template Contaminantiv | % Reads Mapped to Oligonucleotides | % Reads Involving Probev |
|---|---|---|---|---|---|
| N1_pc (n = 1) | EUA-kit | 96% | nd | 4% | <1% |
| N1_fp (n = 2) | EUA-kit | nd | 34% (0%) | 66% (0%) | <1% (0%) |
| N3_fp (n = 2) | pre-EUA | nd | nd | 98% (1%) | 51% (2%) |
| N3_pc (n = 1) | EUA-kit | 42% | nd | 58% | <1% |
| N3_fp (n = 14) | EUA-kit | nd | nd | >99% (0%) | 37% (4%) |
| N3_fp (n = 6) | Commercial | nd | nd | 94% (6%) | 43% (10%) |
nd: not detected.
i) pc: positive control; fp: false-positive reactivity during RT-PCR.
ii) EUA-kit: Emergency Use Authorization (first lot of CDC distributed kits); pre-EUA: an aliquot of the materials from the internal CLIA validation of the assay; Commercial: components ordered from a commercial supplier.
iii) reference is the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel positive control which is derived from the Wuhan-Hu-1 sequence (GenBank accession number MN908947).
iv) synthetic template produced by CDC at approximately the same time as kit production containing differentiating bases (Fig 1).
v) the percent of merged reads that contained partial or complete probe sequences and therefore could contribute to false reactive signal during RT-PCR. Due to library preparation, the proportion of NGS reads in each category may not accurately reflect the proportion of product in the RT-PCR output.
The first lot of CDC N1 EUA components was contaminated with a synthetic template (see also Fig 1; S1a and S1b Fig). The N3 components form multimeric molecules involving the probe, leading to fluorescence in the absence of template (Fig 2b). The latter was consistent regardless of the source of oligonucleotides tested. Mean values with standard deviation in parentheses shown for all results with n > 1.