Dataset Information

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction.

ABSTRACT: The genetic identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is based on viral RNA extraction prior to RT-qPCR assay. However, recent studies have supported the elimination of the extraction step. This study was performed to assess the necessity for the RNA extraction, by comparing the efficacy of RT-qPCR in several direct approaches versus the gold standard RNA extraction, in the detection of SARS-CoV-2 in laboratory samples, as well as in clinical oro-nasopharyngeal SARS-CoV-2 swabs. The findings showed an advantage for the extraction procedure; however a direct no-buffer approach might be an alternative, since it identified more than 60% of positive clinical specimens.

SUBMITTER: Israeli O

PROVIDER: S-EPMC7416699 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction.

Israeli Ofir O Beth-Din Adi A Paran Nir N Stein Dana D Lazar Shirley S Weiss Shay S Milrot Elad E Atiya-Nasagi Yafit Y Yitzhaki Shmuel S Laskar Orly O Schuster Ofir O

International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 20200810

The genetic identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is based on viral RNA extraction prior to RT-qPCR assay. However, recent studies have supported the elimination of the extraction step. This study was performed to assess the necessity for the RNA extraction, by comparing the efficacy of RT-qPCR in several direct approaches versus the gold standard RNA extraction, in the detection of SARS-CoV-2 in laboratory samples, as well as in clinical oro-nasopharynge ...[more]

PMID: 32791207

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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

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction.

Publications

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction.

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