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. 2020 Dec 17;59(1):e02369-20.
doi: 10.1128/JCM.02369-20. Print 2020 Dec 17.

Identification of a Polymorphism in the N Gene of SARS-CoV-2 That Adversely Impacts Detection by Reverse Transcription-PCR

Manu Vanaerschot  1 , Sabrina A Mann  1   2 , James T Webber  1 , Jack Kamm  1 , Sidney M Bell  3 , John Bell  4 , Si Noon Hong  5 , Minh Phuong Nguyen  5 , Lienna Y Chan  1 , Karan D Bhatt  1 , Michelle Tan  1 , Angela M Detweiler  1 , Alex Espinosa  4 , Wesley Wu  1 , Joshua Batson  1 , David Dynerman  1 , Debra A Wadford  4 , Andreas S Puschnik  1 , Norma Neff  1 , Vida Ahyong  1 , Steve Miller  2 , Patrick Ayscue  1 , Cristina M Tato  1 , Simon Paul  5 , Amy L Kistler  1 , Joseph L DeRisi  1   2 , Emily D Crawford  6   2
Affiliations

Identification of a Polymorphism in the N Gene of SARS-CoV-2 That Adversely Impacts Detection by Reverse Transcription-PCR

Manu Vanaerschot et al. J Clin Microbiol. .
No abstract available

Keywords: COVID-19; PCR; SARS-CoV-2; diagnosis; mutation; polymorphism; sensitivity.

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Figures

FIG 1
FIG 1
Single mutation in forward N-gene primer binding site, prevalent across the world, decreased SARS-CoV-2 RT-PCR sensitivity. (A) Potential SARS-CoV-2 mutants were identified by their increased ΔCT between the N- and E-gene assays (>2.5× standard deviation of average ΔCT; cutoff indicated by black lines). Dotted lines indicate the average CT value at the limit of detection (LOD) of each assay, above which more variation is expected. NOTD, not detected. (B) Diagram showing a fragment of the N gene, with the N-gene primers and probe originally developed by the National Institute of Infectious Diseases (NIID) in Tokyo, Japan (4), and the identified G29140U mutation indicated. (C) The increased ΔCT(N−E) of mutant lines using the conventional RT-PCR with the wild-type primer was reversed when a primer incorporating the mutation was used. The opposite was observed for wild-type samples that showed an increased ΔCT(N−E) when the mutated primer was used, further validating causality of the G29140U mutation for reduced N-gene RT-PCR performance. Error bars indicate standard errors of the means. ****, significant difference determined by a t test (P < 0.0001). (D) Phylogeny of SARS-CoV-2 isolates with N-gene mutation, including those with the G29140U mutation. Inferred mutation events on the tree are annotated with an asterisk that is colored depending on the allele. Both synonymous variants of the Q289H mutant are found, with the mutation estimated to have recurred 11 times on the tree, and only one of the mutant samples from GISAID was identical by descent to the Madera cluster. One of the wild-type Madera samples was closely related to the mutant cluster, with a common ancestor just before the mutation event. Sequence data are available in Table S1. All code used for analyses and figure generation is described in Text S1.
FIG 1
FIG 1
Single mutation in forward N-gene primer binding site, prevalent across the world, decreased SARS-CoV-2 RT-PCR sensitivity. (A) Potential SARS-CoV-2 mutants were identified by their increased ΔCT between the N- and E-gene assays (>2.5× standard deviation of average ΔCT; cutoff indicated by black lines). Dotted lines indicate the average CT value at the limit of detection (LOD) of each assay, above which more variation is expected. NOTD, not detected. (B) Diagram showing a fragment of the N gene, with the N-gene primers and probe originally developed by the National Institute of Infectious Diseases (NIID) in Tokyo, Japan (4), and the identified G29140U mutation indicated. (C) The increased ΔCT(N−E) of mutant lines using the conventional RT-PCR with the wild-type primer was reversed when a primer incorporating the mutation was used. The opposite was observed for wild-type samples that showed an increased ΔCT(N−E) when the mutated primer was used, further validating causality of the G29140U mutation for reduced N-gene RT-PCR performance. Error bars indicate standard errors of the means. ****, significant difference determined by a t test (P < 0.0001). (D) Phylogeny of SARS-CoV-2 isolates with N-gene mutation, including those with the G29140U mutation. Inferred mutation events on the tree are annotated with an asterisk that is colored depending on the allele. Both synonymous variants of the Q289H mutant are found, with the mutation estimated to have recurred 11 times on the tree, and only one of the mutant samples from GISAID was identical by descent to the Madera cluster. One of the wild-type Madera samples was closely related to the mutant cluster, with a common ancestor just before the mutation event. Sequence data are available in Table S1. All code used for analyses and figure generation is described in Text S1.
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References

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