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Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

ABSTRACT: BACKGROUND:Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES:This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN:RNA extracted from pharyngeal swabs was tested by variplex™?RT-LAMP and Corman's LightMix™?E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™?without RNA extraction and tested in parallel with the Allplex™?and VIASURE BD MAX?RT-PCRs. RESULTS:Using isolated RNA variplex™?RT-LAMP showed a sensitivity of 75 % compared to LightMix?E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™?RT-LAMP conducted on unprocessed samples and Allplex™?and VIASURE RT-PCRs (Cohen's ??ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3 % for variplex™, 84.2 % for Allplex™?and 68.4 % for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100 %. CONCLUSION:The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

SUBMITTER: Rodel J

PROVIDER: S-EPMC7457909 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

Rödel Jürgen J Egerer Renate R Suleyman Aynur A Sommer-Schmid Beatrice B Baier Michael M Henke Andreas A Edel Birgit B Löffler Bettina B

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20200831

<h4>Background</h4>Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction.<h4>Objectives</h4>This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs.<h4>Study design</h4>RNA extra ...[more]

PMID: 32891938

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Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

Dataset Information

Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

Publications

Use of the variplex™ SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis.

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