Emergence of a mutation in the nucleocapsid gene of SARS-CoV-2 interferes with PCR detection in Canada

Abstract

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic ‘blind spots’ for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test

“…In an effort to improve assay performance against emerging SARS-CoV-2 variants, the PLUS combination test introduces an additional SARS-CoV-2 target (RdRp), as this region has been unmodified in all circulating variants of concern (te Velthuis et al 2009;Pachetti et al 2020;Peng et al 2020), and has introduced redesigned N2 oligos for better coverage of circulating mutations that caused N-gene target dropout (Foster et al 2022;Isabel et al 2022). Here, we investigated the performance characteristics of the PLUS combination test, comparing it to the SARS-CoV-2/Flu/RSV combination test authorized for use in Canada since January 2021.…”

“…As SARS-CoV-2 variants of concern continue to emerge worldwide, Cepheid developed a new formulation of their Xpert SARS-CoV-2/Flu/RSV combination test, the Xpert SARS-CoV-2/Flu/RSV PLUS combination test (referred to hereafter as the PLUS combination test). The PLUS combination test has an additional SARS-CoV-2 target, RNA-dependent RNA polymerase (RdRp), to help improve assay robustness against novel SARS-CoV-2 variants and redesigned N2 oligos for better coverage of nucleocapsid mutations in circulation that have been shown to cause N-gene detection dropout using GeneXpert SARS-CoV-2 assays (Foster et al 2022;Isabel et al 2022). RdRp (also known as nsp-12) is the catalytic subunit of multi-subunit polymerase complex that, along with nsp-7 and nsp-8, forms the minimal core components for SARS-CoV-2 RNA synthesis (Peng et al 2020).…”

SARS-CoV-2 viral variants can rapidly be identified for clinical decision making and population surveillance using a high-throughput digital droplet PCR assay

“…Therefore, the lack of amplification is used to make inferences of a non-amplified sequence. The dropout of certain PCR targets has been the basis for monitoring of variant distributions at a global scale 29 – 33 . To unambiguously assess mutated versus wildtype sequences, one could include separate primer/probe sets for wild type and mutated sequences, as has been done previously by others 5 , 20 .…”

Sensitive detection of SARS‐CoV‐2 main protease 3CLpro with an engineered ribonuclease zymogen

“…For example, the appearance of the Omicron SARS‐CoV‐2 variant led to a days‐long lag between the detection of viral infection by RADT versus PCR (Adamson et al, 2022). Even PCR‐based testing, which is the gold standard, has lost sensitivity due to “gene dropout” in the spike and nucleocapsid proteins (Ippoliti et al, 2022; Isabel et al, 2022; Robles‐Escajeda et al, 2023).…”