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. 2021 May 27;22(11):5743.
doi: 10.3390/ijms22115743.

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

Igor P Oscorbin  1 , Georgiy Yu Shevelev  1 , Ksenia A Pronyaeva  1 , Andrey A Stepanov  1 , Darya V Shamovskaya  1 , Olga V Mishukova  1 , Dmitrii V Pyshnyi  1 , Maksim L Filipenko  1
Affiliations

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

Igor P Oscorbin et al. Int J Mol Sci. .

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.

Keywords: LAMP; SARS-CoV-2; coronavirus; loop-mediated isothermal amplification; melting curve analysis; multiplex amplification.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(a) LAMP with plasmids and different primers for SARS-CoV-2 and MS2; (b) LAMP with standard plasmids, monoplexes SARS-CoV-2, MS2 and duplex primer set MS2-SARS-CoV-2-E. Each primer set is marked by the color specified in the legend. Time-to-threshold (Tt) values are presented on the X-axis, and the amount of template per reaction on the Y-axis. Each run was triplicated; error bars represent one SD.
Figure 2
Figure 2
Melting curve analysis of the LAMP products. The graph represents characteristic melting peaks for LAMP products obtained with (A) monoplexes CoV-2-E (pink curves) and MS2 (blue curves); (B) duplex MS2-CoV-2-E, where green curves correspond to MS2 and purple curves to CoV-2-E.
Figure 3
Figure 3
LAMP with plasmids and different concentrations of MS2 primers in (a) monoplex, (b) duplex, (d) duplex with MS2 RNA and (c) without it. Each MS2 primer concentration is marked by the color specified in the legend. Tt values are presented on the X-axis, and amount of template per reaction on the Y-axis. Each run was triplicated; error bars represent one SD.
Figure 4
Figure 4
LAMP with CoV-2-E monoplex, duplex and various MS2 RNA concentrations. Each MS2 RNA concentration is marked by the color specified in the legend. Tt values are presented on the X-axis, and the amount of MS2 RNA per reaction on the Y-axis. Each run was triplicated; error bars represent one SD.
Figure 5
Figure 5
The melting curve analysis of the LAMP products: evaluation of the limit of detection for the MS2-CoV-2 duplex. The graph represents characteristic melting peaks for LAMP products obtained with the CoV-2-E monoplex and CoV-2-E RNA template (green curve), with the MS2 monoplex and MS2 RNA template (orange curve), with the MS2-CoV-2-E duplex, 12,000 copies/reaction MS2 RNA template, and with CoV-2-E RNA: 100 copies (purple curve), 50 copies (red curve) and 20 copies (blue curve).
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Supplementary concepts