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

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.

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

“…As it happens for PCR reaction, fluorescence-producing reactions are an important route for detecting these products of amplification optically, both in real-time ( Ganguli et al., 2020 ; Alekseenko et al., 2021 ) and in end-point ( Alekseenko et al., 2021 ; Sherrill-Mix et al., 2021 ) contexts. The mechanisms that lead to fluorescence production include intercalating dyes ( Alekseenko et al., 2021 ), specific oligonucleotide probes ( Jang et al., 2021 ; Oscorbin et al., 2021 ), or enzymatic reactions that culminate in the production of fluorescence ( Joung et al., 2020 ; Sun et al., 2021 ). Intercalating dyes, such as SYBR Green ( Alekseenko et al., 2021 ) or EvaGreen ( Alekseenko et al., 2021 ), are nonspecific but can be used for both real-time monitoring, or end-point measuring of amplification.…”

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

“…The work by Oscorbin et al demonstrates the ability to detect a duplex of bacteriophage MS2 RNA and the E gene of SARS-CoV-2 using intercalating dye SYTO-82. After LAMP amplification of these targets of interest and the addition of SYTO-82, a melting curve analysis was performed and demonstrated an exceptionally low LOD for the E gene (20 copies/reaction) in the duplex detection with MS2 [ 89 ]. The melting curve from this study is shown in Figure 4 c and demonstrates that multiplex detection can be achieved without any specificity of the dye.…”

Use of MALDI-TOF mass spectrometry for virus identification: a review

“…8 Kudr et al 4 demonstrated the potential use of electrochemical biosensors for virus identification in their review. In many studies, loop-mediated isothermal amplification (LAMP) [9][10][11][12] and highly multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection [13][14][15][16] have been investigated as potential tools for SARS-CoV-2 detection. In addition to the above-mentioned diagnostic approaches, matrix-assisted laser desorption and ionisation with time-of-flight analyser mass spectrometry (MALDI-TOF MS) for SARS-CoV-2 detection was also demonstrated as another promising alternative in recent studies.…”