Neglected SARS-CoV-2 variants and potential concerns for molecular diagnostics: a framework for nucleic acid amplification test target site quality assurance

Abstract

Molecular tests like polymerase chain reaction were widely used during the COVID-19 pandemic but as the pandemic evolved, so did SARS-CoV-2. This virus acquired mutations, prompting concerns that mutations could compromise molecular test results and be falsely negative. While some manufacturers may have in-house programs for monitoring mutations that could impact their assay performance, it is important to promptly report mutations in circulating viral strains that could adversely impact a diagnostic test result. However, commercial test target sites are proprietary, making independent monitoring difficult. In this study, SARS-CoV-2 test target sites were sequenced to monitor and assess mutations impact, and 29 novel mutations impacting SARS-CoV-2 detection were identified. This framework for molecular test target site quality assurance could be adapted to any molecular test, ensuring accurate diagnostic test results and disease diagnoses.

Keywords: COVID-19; NAAT; PCR; SARS-CoV-2; genome; mutation; quality; sequence; target; variant.

Fig 1

Framework for commercial NAAT target site quality assurance. Solid lines are ideal pathways, dotted lines are possible but inefficient pathways, and dashed lines are practical solutions. Abbreviations: NGS, next-generation sequencing; NAAT, nucleic acid amplification tests.

Fig 2

Dynamic range of the Xpert E and N2 gene targets. Ten-fold serial dilution of synthetic DNA harboring the wild-type E gene target sequence as a calibrator, and one of the following N2 target sequences: (A) wild-type; (B) a N2 G29195T mutant shown to reduce N2 target sensitivity; and (C) a C29200T mutation known to cause N2 target detection failure. Abbreviation: Ct, threshold cycle.

Fig 3

Dual target synthetic DNA-based mutation impact assessment. The principle of mutation impact assessment is illustrated using the Xpert N2 target site as an example. (A) Custom synthetic DNAs composed of a calibrator sequence that remains unchanged (e.g., Xpert E target) and either a wild-type or mutated target sequences (e.g., Xpert N2 target) are compared by NAAT testing following concentration normalization. The average Ct difference between the calibrator and wild-type target (i.e., expected ∆Ct) can be subtracted from those obtained with the calibrator and mutated targets (i.e., observed ∆Ct). (B) When plotted, target site mutations with no impact fall between ±3 SD of the average expected difference between wild-type calibrator and targets; those causing reduced sensitivity are above +3 SD; and mutations resulting in a target detection failure (target Ct = 0.0) result in negative values near −25, given synthetic DNAs are initially normalized to Ct values between 20 and 25. Abbreviations: cycle threshold (Ct) and nucleic acid amplification test (NAAT).