ABSTRACT
Background: Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). Since September 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific, Waltham, MA, USA) has been used to identify viral strains of the new lineage B.1.1.7, since it previously failed to detect the S-gene 69/70 deletion. Rapid detection of these variants of concern can help to contain them and prevent widely spreading throughout the population. This study evaluated S-gene mutations screening with a commercially available qualitative real-time PCR (RT-PCR) assay to detect likely variant of SARS-CoV-2 by the S gene amplification curve non-sigmoidal fluorescence profile. Method(s): The viral RNA of the samples was extracted using the Seegene STARlet automated system combined with StarMag 96x4 Viral DNA/RNA 200C. All samples were subjected to Real Time RT-PCR with the AllplexTM SARS-CoV-2 Assay kit (Seegene Inc, Seul, South Korea) for the qualitative detection of four target genes: E, N, RdRp, and S genes. PCR has been performed by a CFX-96 real-time thermocycler (Bio-Rad Laboratories Inc, Hercules, CA, USA) and Ct values were acquired from the fluorescence channels by the fluorophores FAM (E gene), Quasar 670 (N gene), Cal Red 610 (RdRp and S genes), and HEX (internal control). Gene target amplification curve analysis was performed using Seegene Viewer ver 3.24 (Seegene). The samples tested positive for SARS-CoV-2 viral genome, which presented abnormalities in the fluorescence profile of the amplification curve of the RdRP/S genes, were further subjected to the amplification protocol by the GSD NovaTYpe SARS-CoV-2 amplification kit (Nova Tec Immunodiagnostica, Dietzenbach, Hessen, Germany), and to definitively confirm the presence of the English variant (lineage B.1.1.7), subsequently subjected to Next Generation Sequencing (NGS). Result(s): Thirty respiratory samples subjected to amplification using the AllplexTM SARS-CoV-2 Assay in early January 2021, showed a reduction in amplification efficiency on the fluorescence profile of RdRP/S genes with slope and inflection point variations. The profile of the SARS-CoV-2 English variant (lineage B.1.1.7) for the 30 samples, was performed first by qualitative test in RT-PCR, GSD Nova TYpe SARS-CoV-2, and subsequently confirmed by NGS technology (analysis performed in an external laboratory). Both analytical methodologies identified all 30 samples with the B.1.1.7. lineage of SARS-CoV-2. This last diagnostic proof pushed our working group to evaluate the presence and degree of spread of the English variant in the area of the Napoli 3 Sud ASL, testing the viral genome of 900 samples to RT-PCR using the Allplex TM SARS-Cov-2 kit, alterations in the fluorescence profile of the amplification curves related to the S gene were observed and confirmed using the GSD NovaTYpe SARS-CoV-2 kit. Conclusion(s): Since late 2020, several variants of SARS-CoV-2 have emerged around the world. The gold standard molecular method to detect a specific variant consists in the total or partial sequencing of the virus genome, but today the latter remains expensive and time-consuming, limiting its implementation in all diagnostic samples. PCR-based screening approaches are relatively cheaper, and results can be verified in a few hours. The indirect approach in RT-PCR, on SARS-Cov-2 diagnostic tests of positive samples, of the non-sigmoidal fluorescence profile of the S gene using AllplexTM SARS-CoV-2 PCR assay allows a quick and cheaper prediction of the lineage B.1.1.7. Therefore, using Allplex SARS-COV 2 can be used as an early and first-line screening, before eventually using the sequencing of the viral genome. Copyright © 2022 EDIZIONI MINERVA MEDICA.