DETECTION OF A SARS-COV2 RNA USING MULTIPLEX REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION WITH MELTING CURVE ANALYSIS

DOI: https://doi.org/10.29296/25877313-2020-12-01
Issue: 
12
Year: 
2020

I.P. Oscorbin Ph.D. (Biol.), Minor Researcher, Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk) G.Yu. Shevelev Ph.D. (Chem.), Head of Laboratory, Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk) E-mail: osc.igor@gmail.com K. A. Pronyaeva Laboratory Assistant, Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk) A.A. Stepanov Ph.D. (Med.), Head of Laboratory, Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk) D.V. Pyshny Dr.Sc. (Chem.), Corr. Member of RAS, Director of the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk) M.L. Filipenko Ph.D. (Biol.), Head of Laboratory, Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk)

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 loop-mediated 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 multi-plex 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 detec-tion 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 re-sults was 92%. Conclusion. We have developed a prototype of a multiplex LAMP-based test system for a detecting SARS-CoV2 coronavirus RNA. The developed ap-proach can be used as an alternative to RT-PCR in diagnostic practice for saving of machine and personnel time.

Keywords: 
SARS-CoV2
coronavirus
isothermal loop amplification
LAMP
multiplex amplification

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