key: cord-0875353-n3wwsbev authors: Hasan, Mohammad Rubayet; Sundararaju, Sathyavathi; Manickam, Chidambaram; Mirza, Faheem; Al-Hail, Hamad; Lorenz, Stephan; Tang, Patrick title: A Novel Point Mutation in the N Gene of SARS-CoV-2 May Affect the Detection of the Virus by Reverse Transcription-Quantitative PCR date: 2021-03-19 journal: J Clin Microbiol DOI: 10.1128/jcm.03278-20 sha: b4c7b42b87f495407ce957d35f7a2cf9e55a0cbd doc_id: 875353 cord_uid: n3wwsbev Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, laboratory testing to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription PCR (RT-qPCR) has played a central role in mitigating the spread of the virus (1). Soon after the viral genome sequences were available, several RT-qPCR assays were developed and made available by World Health Organization (WHO) for public use (https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf). The primer and probe sequences for these assays were chosen from multiple target genes within the viral genome such as the E gene, RdRp gene, ORF1ab and N gene. Many commercial and laboratory-developed assays were developed for SARS-CoV-2 detection based on these primer and probe sequences. The large-scale sustained person-to-person transmission of SARS-CoV-2 has led to many mutational events, some of which may affect the sensitivity and specificity of available PCR assays (2). Recently, mutations in the E gene (C26340T) and N gene (C29200T) were reported affecting the detection of target genes by two commercial assays in 8 and 1 patients, respectively. Interestingly, both mutations are of C>T type, a common single nucleotide polymorphism (SNP) that may be associated with strong host cell mRNA editing mechanisms known as APOBEC cytidine deaminase (3, 4). Another study found a G to U substitution in position 29140 that affected the sensitivity of detection of N gene-based assays (5). Here we report a novel N gene mutation (C29200A) seen in 3 patients, which affected the detection of SARS-CoV-2 N gene by a commercial assay. use of anonymous, residual pathological specimens that fall under the category "exempted" by the Sidra Medicine Institutional Review Board. We sequenced the entire N gene in these samples, along with two other samples that were positive for the N gene and E gene, by Sanger sequencing. cDNA was prepared using the Superscript IV RT cDNA synthesis kit (ThermoFisher). The N gene was amplified with 3 sets of primers (see Table S1 in the supplemental material) using Platinum Taq high-fidelity DNA polymerase (ThermoFisher). Sequencing was performed with a BigDye Terminator v3.1 cycle sequencing kit (ThermoFisher). All procedures were performed according to manufacturer's instructions. Sequence data were analyzed in Geneious prime 2019.2.3. Sequencing was unsuccessful for 1 of the 4 suspected N gene variants. A point mutation (C29200A) was consistently detected in all three suspected variants but not in the SARS-CoV-2 consensus sequence ( Fig. 1) . A review of different SARS-CoV-2 target sequences that were recommended by the WHO revealed that the point mutation falls into the probe sequence of one of the CDC assays (2019_nCoV_N2) (https://www.who.int/docs/ default-source/coronaviruse/whoinhouseassays.pdf). Although the N gene target of the Cepheid Xpert assay is proprietary, the mutation in one of the CDC-developed assay targets suggest that a C29200A substitution may be responsible for the failure of amplification of the N gene by Xpert. Notably, none of these results were reported as false negative by the test platform because the E gene was amplified, and a discrepancy in the results for two targets warranted further investigation. Interestingly, a point mutation in the same genome position was independently reported from Germany in another study, but with a C29200T substitution. According to the study, 0.2% of the isolates in the EpiCoV database contain the C29200T mutation (4). However, using a BLAST search against the entire nucleotide collection in the NCBI, we were unable to find any reports of the C29200A mutation. Although only a small fraction of our SARS-CoV-2 isolates harbored this mutation, detection of the point mutation at 4 time points independent of each other suggests that there may be more cases in the community that remain to be detected. Apart from the Xpert assay, the C29200A mutation may also affect the performance of other commercial or laboratory-developed RT-qPCR tests for SARS-CoV-2 that are based on the same set of primers and probe used for the CDC's 2019_nCoV_N2 assay. Our results provide further support for the possibility that novel SARS-CoV-2 variants may escape detection by nucleic acid amplification tests (NAAT), in particular if they are tested by assays based on single gene targets, and attest to the importance of having more than one PCR assay available for confirmation of ambiguous results. Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.1 MB. Mutations on COVID-19 diagnostic targets 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 dual-target diagnostic assay SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by reverse transcription-PCR We are grateful for the efforts of all technologists in the Molecular Infectious Disease Laboratory at Sidra Medicine.