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A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

ABSTRACT: Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

SUBMITTER: Fuchs Wightman F

PROVIDER: S-EPMC8174584 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Fuchs Wightman Federico F Godoy Herz Micaela A MA Muñoz Juan C JC Stigliano José N JN Bragado Laureano L Moreno Nicolas Nieto NN Palavecino Marcos M Servi Lucas L Cabrerizo Gonzalo G Clemente José J Avaro Martín M Pontoriero Andrea A Benedetti Estefanía E Baumeister Elsa E Rudolf Fabian F Remes Lenicov Federico F Garcia Cybele C Buggiano Valeria V Kornblihtt Alberto R AR Srebrow Anabella A de la Mata Manuel M Muñoz Manuel J MJ Schor Ignacio E IE Petrillo Ezequiel E

RNA biology 20210526 12

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents a ...[more]

PMID: 33966602

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Project description:Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants.ImportanceWe have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

Project description:BackgroundSensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines.MethodsHere, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here.ResultsMost RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost.ConclusionsWith extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

Dataset Information

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Publications

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

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