Detection of a SARS-CoV2 RNA using multiplex reverse transcription loop-mediated isothermal amplification with melting curve analysis

Relevance. Ongoing COVID-19 pandemic caused by the SARS-CoV2 coronavirus urges the need for diagnostic tests to detect the RNA of the SARS-CoV2 virus. The most common method, RT-PCR, allows to obtain the test results within 1.5-2 hours. However, the lack of capacity of diagnostic laboratories necessitates a developing of more rapid testing methods. Purpose of the study. The development of a method for a detection of SARS-CoV2 RNA using multiplex reverse transcription loopmediated isothermal amplification with melting curve analysis Material and methods. The constructed plasmids with a fragment of the SARS-CoV2 genome and MS2 phage, fragments of the SARS-CoV2 genomic RNA and MS2 phage were used as standard samples. Clinical samples (nasopharyngeal swabs) were obtained from patients CNMT of ICBFM SB RAS. RNA was isolated using a «RIBO-prep» kit (Central research Institute of Epidemiology (Moscow; Russia)). LAMP was performed in a CFX96 thermocycler (Bio-Rad; USA). Analytical characteristics of LAMP were evaluated using dilutions of standard samples. The clinical sensitivity and specificity of multiplex LAMP were evaluated by testing clinical samples simultaneously with LAMP and RT-PCR. Results. Primers were selected and conditions were optimized for a LAMP-based detection of SARS-CoV2 RNA and MS2 phage RNA, the latter served as an internal control of RNA isolation and amplification. Multiplexing was based on a melting curves analysis of amplification products. The limit of detection of the multiplex LAMP was 20 copies of SARS-CoV2 RNA per reaction. The concordance with real-time RT-PCR of the 40 clinical samples testing results was 92%. Conclusion. We have developed a prototype of a multiplex LAMP-based test system for a detecting SARS-CoV2 coronavirus RNA. The developed approach can be used as an alternative to RT-PCR in diagnostic practice for saving of machine and personnel time.

SARS-CoV2, LAMP, SARS-CoV2, coronavirus, isothermal loop amplification, LAMP, multiplex amplification