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Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including a...

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Autores principales: Oscorbin, Igor P., Shevelev, Georgiy Yu., Pronyaeva, Ksenia A., Stepanov, Andrey A., Shamovskaya, Darya V., Mishukova, Olga V., Pyshnyi, Dmitrii V., Filipenko, Maksim L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197939/
https://www.ncbi.nlm.nih.gov/pubmed/34072209
http://dx.doi.org/10.3390/ijms22115743
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author Oscorbin, Igor P.
Shevelev, Georgiy Yu.
Pronyaeva, Ksenia A.
Stepanov, Andrey A.
Shamovskaya, Darya V.
Mishukova, Olga V.
Pyshnyi, Dmitrii V.
Filipenko, Maksim L.
author_facet Oscorbin, Igor P.
Shevelev, Georgiy Yu.
Pronyaeva, Ksenia A.
Stepanov, Andrey A.
Shamovskaya, Darya V.
Mishukova, Olga V.
Pyshnyi, Dmitrii V.
Filipenko, Maksim L.
author_sort Oscorbin, Igor P.
collection PubMed
description Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.
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spelling pubmed-81979392021-06-14 Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis Oscorbin, Igor P. Shevelev, Georgiy Yu. Pronyaeva, Ksenia A. Stepanov, Andrey A. Shamovskaya, Darya V. Mishukova, Olga V. Pyshnyi, Dmitrii V. Filipenko, Maksim L. Int J Mol Sci Article Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time. MDPI 2021-05-27 /pmc/articles/PMC8197939/ /pubmed/34072209 http://dx.doi.org/10.3390/ijms22115743 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Oscorbin, Igor P.
Shevelev, Georgiy Yu.
Pronyaeva, Ksenia A.
Stepanov, Andrey A.
Shamovskaya, Darya V.
Mishukova, Olga V.
Pyshnyi, Dmitrii V.
Filipenko, Maksim L.
Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title_full Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title_fullStr Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title_full_unstemmed Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title_short Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
title_sort detection of sars-cov-2 rna by a multiplex reverse-transcription loop-mediated isothermal amplification coupled with melting curves analysis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197939/
https://www.ncbi.nlm.nih.gov/pubmed/34072209
http://dx.doi.org/10.3390/ijms22115743
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