Dataset Information

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

ABSTRACT:

Background

Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed.

Objectives

To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials.

Material and methods

Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene.

Results

The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 ?l of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing.

Conclusion

Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

SUBMITTER: Lubke N

PROVIDER: S-EPMC7405857 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Publications

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

Lübke Nadine N Senff Tina T Scherger Sara S Hauka Sandra S Andrée Marcel M Adams Ortwin O Timm Jörg J Walker Andreas A

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20200805

<h4>Background</h4>Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed.<h4>Objectives</h4>To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials.<h4>Material and methods</h4>Different SARS-CoV- ...[more]

PMID: 32795959

Similar Datasets

Project description:ObjectivesWe explore the importance of SARS-CoV-2 sentinel surveillance testing in primary care during a regional COVID-19 outbreak in Austria.DesignProspective cohort study.SettingA single sentinel practice serving 22 829 people in the ski-resort of Schladming-Dachstein.ParticipantsAll 73 patients presenting with mild-to-moderate flu-like symptoms between 24 February and 03 April, 2020.InterventionNasopharyngeal sampling to detect SARS-CoV-2 using real-time reverse transcriptase-quantitative PCR (RT-qPCR).Outcome measuresWe compared RT-qPCR at presentation with confirmed antibody status. We split the outbreak in two parts, by halving the period from the first to the last case, to characterise three cohorts of patients with confirmed infection: early acute (RT-qPCR reactive) in the first half; and late acute (reactive) and late convalescent (non-reactive) in the second half. For each cohort, we report the number of cases detected, the accuracy of RT-qPCR, the duration and variety of symptoms, and the number of viral clades present.ResultsTwenty-two patients were diagnosed with COVID-19 (eight early acute, seven late acute and seven late convalescent), 44 patients tested SARS-CoV-2 negative and 7 were excluded. The sensitivity of RT-qPCR was 100% among all acute cases, dropping to 68.1% when including convalescent. Test specificity was 100%. Mean duration of symptoms for each group were 2 days (range 1-4) among early acute, 4.4 days (1-7) among late acute and 8 days (2-12) among late convalescent. Confirmed infection was associated with loss of taste. Acute infection was associated with loss of taste, nausea/vomiting, breathlessness, sore throat and myalgia; but not anosmia, fever or cough. Transmission clusters of three viral clades (G, GR and L) were identified.ConclusionsRT-qPCR testing in primary care can rapidly and accurately detect SARS-CoV-2 among people with flu-like illness in a heterogeneous viral outbreak. Targeted testing in primary care can support national sentinel surveillance of COVID-19.

Dataset Information

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

Background

Objectives

Material and methods

Results

Conclusion

Publications

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

Similar Datasets

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