Diagnostic pre-screening method based on N-gene dropout or delay to increase feasibility of SARS-CoV-2 VOC B.1.1.7 detection

Abstract

Objectives : To compare the RT-PCR Allplex SARS-CoV-2/FluA/FluB/RSV Assay (Allplex assay) with other methods of detection of VOC B.1.1.7. Methods : Suspected and non-suspected cases of VOC B.1.1.7 were defined according to the VirSNiP assay, which detects N501Y and deletion H69-V70. For pre-screening, the Allplex™ and TaqPath assays were used. Results : One hundred and sixteen suspected and 113 non-suspected cases were included. In the suspected cases, the Allplex assay showed N-gene dropout, or delayed Ct values of 6.27±1.21 and 6.66±1.41 compared with those of the RdRP and S-gene target, respectively. Agreement between the Allplex and TaqPath assays was 100% when the RdRP and S-gene targets had Ct values <35. Agreement between the Allplex and VirSNiP assays was 100% with Ct value <30. Conclusions : The Allplex assay showed excellent agreement with the current pre-screening method for VOC B.1.1.7. In addition, its automated processing enhances the feasibility of widespread use in laboratories.

Rapid Detection of the Omicron (B.1.1.529) SARS-CoV-2 Variant Using a COVID-19 Diagnostic PCR Assay

“…Additionally, the Omicron sublineage BA.2 (not carrying the del69/70 mutation) does not show S-gene dropout ( 7 ). Thus, as for Alpha or Delta variants ( 9 – 11 ), N-gene dropout, which may be qualified as a shift in the N-gene cycle threshold ( C T ) value compared to other target genes’ values, in multiplex PCR assays could be a valuable indicator of the Omicron variant. Consistently, Omicron variant-defining mutations, such as P13L, E31del, R32del, S33del, R203K, and G204R in the N protein, could lead to N-gene target failure in PCR assays that target the N gene.…”

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

“…Even more concerning is the emergence of such mutations amongst SARS-CoV-2 strains designated as VOI's and VOC's. Emergence of partial diagnostic escape mutants is not unique to the Xpert® Xpress SARS-CoV-2 PCR assay as other commercial assays have also been affected [11][12][13][14][15] . Inevitably, diagnostic escape mutations will continue to emerge as SARS-CoV-2 evolves.…”

Use of the Ct difference between the Nucleocapside (N) and the Spike (S) or RNA-dependent RNA polymerase (RdRP) genes as a preliminary screening for SARS-CoV-2 variants with the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay

“…As stated by other authors previously the appearance of either Dropouts or late amplifications at validated commercial assays should serve as a hint on the search for potential new variants ( Kidd et al, 2021 ; Sánchez-Calvo et al, 2021 ). Especially so when the affected regions such as the N gene are known to be more conservated than others( Wollschläger et al, 2021 ; Grifoni et al, 2020 ).…”

“…During the second half of 2021 the Alpha variant is being progressively replaced by the Delta variant in many European countries ( EU-EEA, 2021 ). For Alpha several descriptions of PCR primer compromises have being described, most noticeably the S gene dropout at the Thermofisher assay ( Bal et al, 2021 ) ( Kidd et al, 2021 ), which has also been described for the Omicron variant ( SARS-CoV-2, 2021 ), and the N gene late amplification at the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Seegene, Korea) assay ( Sánchez-Calvo et al, 2021 ; Wollschläger et al, 2021 ). This has not been the case for Delta or any other VOC or VOI.…”