key: cord-0897327-3g78de7k authors: Laine, Pia; Nihtilä, Hanna; Mustanoja, Ella; Lyyski, Annina; Ylinen, Anne; Hurme, Jukka; Paulin, Lars; Jokiranta, Sakari; Auvinen, Petri; Meri, Taru title: SARS‐CoV‐2 variant with mutations in N gene affecting detection by widely used PCR primers date: 2021-11-02 journal: J Med Virol DOI: 10.1002/jmv.27418 sha: ded0ad841bc0fa73a02b1e55d60ebc071fc911ba doc_id: 897327 cord_uid: 3g78de7k While most of the spontaneous mutations in the viral genome have no functional, diagnostic, or clinical consequences, some have. In February 2021, we noticed in Southern Finland coronavirus disease 2019 cases where two commercial polymerase chain reaction (PCR) analyses failed to recognize the used N gene target but recognized the other target gene of severe acute respiratory syndrome coronavirus 2. Complete viral genome sequence analysis of the strains revealed several mutations that were not found at that time in public databases. A short 3 bp deletion and three subsequent single nucleotide polymorphisms in the N gene were found exactly at the site where an early published and widely used N gene‐based PCR primer is located, explaining the negative results in the N gene PCR. Later the variant strain was identified as a member of the B.1.1.318 Pango lineage that had first been found from Nigerian samples collected in January 2021. This strain shares with the Beta variant the S gene E484K mutation linked to impaired vaccine protection, but differs from this variant in several other ways, for example by deletions in the N gene region. Mutations in the N gene causing diagnostic resistance and on the other hand E484K mutation in the causing altered infectivity warrants careful inspection on virus variants that might get underdiagnosed. Currently circulating and emerging SARS-CoV-2 viruses are naturally expanding genetic diversity. SARS-CoV-2 genome has preferences on nucleotide mutation restricted with effect on coding i.e., possible nonsynonymous mutations that are not tolerated at the level of the protein structure/function axis. A to C or T and T to A are the most frequent nucleotide changes. 3 Genome of SARS-CoV-2 has a complicated RNA secondary structure that could restrict allowed mutations to a level that does not affect protein-coding capacity. 4, 5 The golden standard test for diagnosis of COVID-19 is quantitative reverse-transcription polymerase chain reaction (RT-qPCR), which is performed to RNA isolated from a nasopharyngeal swab. Antigen detection and antibody tests are also widely used. 6 The aim of this study was to analyze in detail the genomic features of SARS-CoV-2 variants, which were not identified with two widely used commercially available RT-PCR diagnostic methods. The first nasopharyngeal swab sample for Covid-analysis was taken on 6.2.2021 from a 44-year-old male living in Southern Finland. The patient was symptomatic and hospitalized the following week. The second patient from the same workplace, a 24-year-old male, gave a sample on February 19, 2021. (Table S1) . We also compared the N gene sequence to NC_045512.2 reference and primer sequences (Figure 2 ). Reference sequences were from diagnostic targets designed and suggested by WHO in the beginning of the current pandemic. We noticed that the site for forward primer (http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.htm) contains three mutations at the 5ʹ end and specifically a 3 bp deletion at the 3ʹ end of the primer, which most likely explain our results from the two different PCR-assays. Additional supporting information may be found in the online version of the article at the publisher's website. A new coronavirus associated with human respiratory disease in China Structure of the SARS-CoV-2 spike receptorbinding domain bound to the ACE2 receptor Time series prediction of COVID-19 by mutation rate analysis using recurrent neural network-based LSTM model RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation Current and innovative methods for the diagnosis of COVID19 infection (Review) Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by reverse transcription-PCR Discrepancy between PCR based SARS-CoV-2 tests suggests the need to re-evaluate diagnostic assays SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene A recurrent mutation at position 26340 of SARS-CoV-2 is associated with failure of the E gene quantitative reverse transcription-PCR utilized in a commercial dualtarget diagnostic assay Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome The authors declare that there are no conflict of interests. Pia Laine, designed sequencing strategy, analyzed sequence data, and wrote manuscript. Hanna Nihtilä, analyzed patient samples and sequence data, prepared figures, and wrote manuscript. Ella Mustanoja, analyzed sequence data, took part into writing process. Annina Lyyski, analyzed sequence data, took part into writing process. Anne Ylinen, analyzed sequence data, and took part into writing process. Jukka Hurme, participated in study design. Lars Paulin, designed sequencing strategy, took part into writing process. Sakari Jokiranta, participated in study design, wrote manuscript. Petri Auvinen, wrote manuscipt. Taru Meri, prepared figures, participated in study design, wrote manuscript. Genome sequence of this variant was deposited to GISAID (accession ID EPI_ISL_1061414).