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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Published inInternational journal of molecular sciences Vol. 22; no. 11; p. 5743
Main Authors Oscorbin, Igor P., Shevelev, Georgiy Yu, Pronyaeva, Ksenia A., Stepanov, Andrey A., Shamovskaya, Darya V., Mishukova, Olga V., Pyshnyi, Dmitrii V., Filipenko, Maksim L.
Format Journal Article
LanguageEnglish
Published MDPI 27.05.2021
MDPI AG
Subjects
coronavirus
LAMP
loop-mediated isothermal amplification
melting curve analysis
multiplex amplification
SARS-CoV-2
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Abstract 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.
AbstractList 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.
Author Pyshnyi, Dmitrii V.
Pronyaeva, Ksenia A.
Oscorbin, Igor P.
Stepanov, Andrey A.
Filipenko, Maksim L.
Shevelev, Georgiy Yu
Shamovskaya, Darya V.
Mishukova, Olga V.
AuthorAffiliation Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8 Lavrentiev Avenue, 630090 Novosibirsk, Russia; metatezis@gmail.com (G.Y.S.); ks_pronyaeva@mail.ru (K.A.P.); stepanov_aa@cnmt.ru (A.A.S.); shamovskaya@gmail.com (D.V.S.); mishukova_olga@inbox.ru (O.V.M.); pyshnyi@niboch.nsc.ru (D.V.P.); mlfilipenko@gmail.com (M.L.F.)
AuthorAffiliation_xml – name: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8 Lavrentiev Avenue, 630090 Novosibirsk, Russia; metatezis@gmail.com (G.Y.S.); ks_pronyaeva@mail.ru (K.A.P.); stepanov_aa@cnmt.ru (A.A.S.); shamovskaya@gmail.com (D.V.S.); mishukova_olga@inbox.ru (O.V.M.); pyshnyi@niboch.nsc.ru (D.V.P.); mlfilipenko@gmail.com (M.L.F.)
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  publication-title: Sci. Rep.
  doi: 10.1038/srep40125
  contributor:
    fullname: Liu
– volume: 129
  start-page: 104446
  year: 2020
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  article-title: Evaluation of rapid diagnosis of novel coronavirus disease (COVID-19) using loop-mediated isothermal amplification
  publication-title: J. Clin. Virol.
  doi: 10.1016/j.jcv.2020.104446
  contributor:
    fullname: Kitagawa
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Snippet Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than...
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SubjectTerms coronavirus
LAMP
loop-mediated isothermal amplification
melting curve analysis
multiplex amplification
SARS-CoV-2
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Title Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis
URI https://search.proquest.com/docview/2536466746
https://pubmed.ncbi.nlm.nih.gov/PMC8197939
https://doaj.org/article/171b8b7138b74ea3a1f5a58cf9844fcb
Volume 22
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