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La Rivista Italiana della Medicina di Laboratorio 2022 Jul 04

DOI: 10.23736/S1825-859X.22.00145-1

Copyright © 2022 EDIZIONI MINERVA MEDICA

language: Italian

Precision molecular diagnostic: a single strategy to investigate SARS-CoV-2 lineage B.1.1.7

Giovanni TRINCHESE , Maria C. DI MARTINO, Angela SARAIELLO

U.O.C. Patologia Clinica, P.O. S. Maria della Pietà, Nola, Napoli, Italia


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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) has been used to identify viral strains of the new lineage B.1.1.7, since it previously failed to detect the S-gene Δ69/70 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.
METHODS: 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 Allplex™ SARS-CoV-2 Assay kit (Seegene) 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) 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), and to definitively confirm the presence of the English variant (lineage B.1.1.7), subsequently subjected to Next Generation Sequencing (NGS).
RESULTS: Thirty respiratory samples subjected to amplification using the Allplex™ 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 ™ 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.
CONCLUSIONS: 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 nonsigmoidal fluorescence profile of the S gene using Allplex™ 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.


KEY WORDS: B.1.1.7; SARS-CoV-2; Allplex™ SARS-CoV-2 Assay; S gene

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