Cargando…
A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test
Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnost...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560768/ https://www.ncbi.nlm.nih.gov/pubmed/34725400 http://dx.doi.org/10.1038/s41598-021-00827-0 |
_version_ | 1784592988145451008 |
---|---|
author | Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Wenzel, Folker Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk |
author_facet | Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Wenzel, Folker Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk |
author_sort | Schmidt, Jonas |
collection | PubMed |
description | Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories. |
format | Online Article Text |
id | pubmed-8560768 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-85607682021-11-03 A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Wenzel, Folker Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk Sci Rep Article Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories. Nature Publishing Group UK 2021-11-01 /pmc/articles/PMC8560768/ /pubmed/34725400 http://dx.doi.org/10.1038/s41598-021-00827-0 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Schmidt, Jonas Berghaus, Sandro Blessing, Frithjof Wenzel, Folker Herbeck, Holger Blessing, Josef Schierack, Peter Rödiger, Stefan Roggenbuck, Dirk A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title | A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title_full | A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title_fullStr | A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title_full_unstemmed | A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title_short | A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test |
title_sort | semi-automated, isolation-free, high-throughput sars-cov-2 reverse transcriptase (rt) loop-mediated isothermal amplification (lamp) test |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560768/ https://www.ncbi.nlm.nih.gov/pubmed/34725400 http://dx.doi.org/10.1038/s41598-021-00827-0 |
work_keys_str_mv | AT schmidtjonas asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT berghaussandro asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT blessingfrithjof asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT wenzelfolker asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT herbeckholger asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT blessingjosef asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT schierackpeter asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT rodigerstefan asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT roggenbuckdirk asemiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT schmidtjonas semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT berghaussandro semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT blessingfrithjof semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT wenzelfolker semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT herbeckholger semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT blessingjosef semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT schierackpeter semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT rodigerstefan semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest AT roggenbuckdirk semiautomatedisolationfreehighthroughputsarscov2reversetranscriptasertloopmediatedisothermalamplificationlamptest |