Precision molecular diagnostic: a single strategy to investigate saRs-cov-2 and lineage b.1.1.7

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.