Dataset Information

Rapid Base-Specific Calling of SARS-CoV-2 Variants of Concern Using Combined RT-PCR Melting Curve Screening and SIRPH Technology.

ABSTRACT:

Background

The emergence of novel variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands fast and reliable detection of such variants in local populations.

Methods

Here we present a cost-efficient and fast workflow combining a prescreening of SARS-CoV-2-positive samples using reverse transcription polymerase chain reaction melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions.

Results

The entire workflow from positive SARS-CoV-2 testing to base-specific identification of variants requires about 24 hours.

Conclusions

We applied the sensitive method to monitor local variant of concern outbreaks in SARS-CoV-2-positive samples collected in a confined region of Germany.

SUBMITTER: Tierling S

PROVIDER: S-EPMC8364759 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid Base-Specific Calling of SARS-CoV-2 Variants of Concern Using Combined RT-PCR Melting Curve Screening and SIRPH Technology.

Tierling Sascha S Kattler Kathrin K Vogelgesang Markus M Pfuhl Thorsten T Lohse Stefan S Lo Porto Christina C Schmitt Beate B Nastasja Seiwert S Salhab Abdulrahman A Smola Sigrun S Walter Jörn J

Open forum infectious diseases 20210714 8

<h4>Background</h4>The emergence of novel variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands fast and reliable detection of such variants in local populations.<h4>Methods</h4>Here we present a cost-efficient and fast workflow combining a prescreening of SARS-CoV-2-positive samples using reverse transcription polymerase chain reaction melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions.<h4>Results</h4>The entire wor ...[more]

PMID: 34409122

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Project description:BACKGROUND: Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. METHODS: In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. RESULTS: A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. CONCLUSION: The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.

Dataset Information

Rapid Base-Specific Calling of SARS-CoV-2 Variants of Concern Using Combined RT-PCR Melting Curve Screening and SIRPH Technology.

Background

Methods

Results

Conclusions

Publications

Rapid Base-Specific Calling of SARS-CoV-2 Variants of Concern Using Combined RT-PCR Melting Curve Screening and SIRPH Technology.

Similar Datasets

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