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Amplification Failure by the Cepheid Xpert Xpress SARS-CoV-2 Assay due to a Single-Nucleotide Variant of the N Gene in the Omicron Variant by Sunghwan Shin, Tae Hyun Um, Chong Rae Cho, Jeonghyun Chang

Background: Rapid identification of SARS-CoV-2 infection using molecular testing has played an important role in preventing the spread of COVID-19. However, the failure of SARS-CoV-2 N gene amplification in the Cepheid Xpert SARS-CoV-2 assay could lead to the failed detection of infections, possibly leading to spread. In this study, we examined N gene amplification failure due to a single-nucleotide variant (SNV) in the N2 region of the gene.
Methods: Xpert assay results obtained at our hospital since March 2021 were retrospectively reviewed and samples with strong E gene and failed N gene amplification were selected. Whole-genome sequencing was performed using the Illumina platform. Lineage analyses were conducted and the N2 target region of the US CDC 2019-nCoV real-time PCR primer sequence, used in PCR assays of SARS-CoV-2 infection, was compared with the reference SARS-COV-2 sequence (Wuhan-Hu-1, NC_045512.2).
Results: The two samples eligible for this study were classified as BA.5.2 (22B, Omicron) and included two synony-mous SNVs, C29197T and C29200T, respectively. Both variants resulted in synonymous mutation of the N gene encoding alanine. The distribution of variants varied across different countries.
Conclusions: Clinical laboratories performing molecular tests targeting the N gene of SARS-CoV-2 should consider the probability of N gene amplification failure when reporting the test results.

DOI: 10.7754/Clin.Lab.2023.230543