Combinations of PCR and Isothermal Amplification Techniques Are Suitable for Fast and Sensitive Detection of SARS-CoV-2 Viral RNA.

Dmitriy A Varlamov,Konstantin B Ignatov,Vladimir M Kramarov,Evgenia V Smirnova,Konstantin A Blagodatskikh

doi:10.3389/fbioe.2020.604793

Abstract

The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of August 31, 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR techniques and can be used to for SARS-CoV-2 detection. The proposed methods based on the modern hybrid amplification techniques markedly improve virus detection and, therefore, can be extremely useful in the development of new diagnostic kits.

Highlights

The SARS-CoV-2 pandemic started in late December 2019, in Wuhan, China (Jiang and Shi, 2020; Wu et al, 2020; Zhou et al, 2020)
The sensitivity of the Reverse transcription (RT)-qPCDR and RT-qPCR techniques in detecting SARS-CoV-2 RNA was evaluated using the two-step assay in which the RT and cDNA amplification steps were performed in two separate reactions
SD DNA polymerase used to amplify the cDNA is a thermostable Taq DNA polymerase mutant that has strong 5 –3 strand displacement and 5 –3 polymerase activities. This polymerase is suitable for polymerase chain reaction (PCR), polymerase chain displacement reaction (PCDR), and isothermal DNA amplifications (Ignatov et al, 2014; Shchit et al, 2017; Smith, 2017; Alyethodi et al, 2018; Lou et al, 2018; Wang et al, 2018)

Summary

Introduction

The SARS-CoV-2 pandemic started in late December 2019, in Wuhan, China (Jiang and Shi, 2020; Wu et al, 2020; Zhou et al, 2020). By August 31, 2020, over 25 million cases of SARS-CoV-2 infection have been confirmed worldwide, with a death toll over 0.8 million. One key aspect of a safe transition from lockdown is the ability to continuously test the population for the presence of SARS-CoV-2 and COVID-19 infected individuals. The SARS-CoV-2 virion contains a single-stranded positivesense RNA genome, 30,000 nucleotides in length (Zhou et al, 2020). Testing involves the reverse transcription (RT) of SARS-CoV-2 RNA into complementary DNA (cDNA), followed by amplification of targeted regions of the cDNA. Standard molecular techniques for the detection of the virus are quantitative RT polymerase chain reaction (RT-qPCR) (Bruce et al, 2020; Chu et al, 2020; Corman et al, 2020; Garafutdinov et al, 2020; Gray et al, 2020; Yip et al, 2020; Zhu et al, 2020) and RT loop-mediated isothermal amplification (RT-LAMP)

Methods

Results

Conclusion