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

• Published in: International 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
• Language: English
• Published: MDPI 27.05.2021; MDPI AG
• Subjects: coronavirus; LAMP; loop-mediated isothermal amplification; melting curve analysis; multiplex amplification; SARS-CoV-2
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22115743

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.