At the end of 2021, we observed an increase in N-gene target failures (NGTF) with the TaqPathTM COVID-19 CE-IVD RT-PCR Kit from Thermo Fisher Scientific (TaqPath). We subsequently used whole-genome sequencing (Oxford Nanopore Technology) to identify potential issues with N-gene PCR efficacy. Among 168,101 positive samples with a cycle threshold (CT) value <30 from August 2021 to May 2022, 194 specimens without N-gene amplification by PCR were identified (0.12%). Most NGTF samples originated from a wave of infection attributable to the Delta variant (B.1.617.2) and its sublineages. Sequencing revealed the nucleotide substitution G28922T (A217S) in 151 samples (88.8%). The substitution G215C, a hallmark mutation for Delta lineages, was concurrently present in all of these samples. Ten samples (5.9%) carried the deletion 28,913-28,918 (del214/215), eight samples (4.7%) the deletion 28,913-28,915 (del214) and one sample (0.6%) the deletion 28,892-28,930 (del207-219). Samples showing intact N-gene amplification by PCR lacked these specific mutations, but delayed-type amplification (i.e., partial or pNGTF) was attributable to the exclusive presence of A217S. As the N gene is a common target in many RT-PCR methods for SARS-CoV-2, an in-depth analysis of single-target failures using a combination with viral whole genome sequencing may allow for the identification of diagnostic flaws and eventual new variants.
Keywords: SARS-CoV-2; polymerase chain reaction; target failure; whole-genome sequencing.