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

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.

Keywords: COVID-19; PCR-LAMP; SARS-CoV-2; hybrid virus detection technique; loop-mediated isothermal amplification; polymerase chain displacement reaction.

FIGURE 1

Comparison of two-step RT-qPCR and RT-qPCDR assays. After reverse transcription (RT) of SARS-CoV-2 RNA, the generated cDNA was detected by qPCR (A) or qPCDR (B). The quantitative real-time reaction assays contained 5000, 500, 50, or 5 copies of cDNA per reaction, or a no template control (NTC). RT-qPCDR provided better sensitivity in the assay than RT-qPCR. All curves reflect the mean relative fluorescence units (RFU) of three replicate reactions.

FIGURE 2

Comparison of one-step RT-qLAMP and RT-q(PCR-LAMP) assays. The reaction assays contained 5000, 500, 50, or 5 copies of SARS-CoV-2 RNA molecules, or a no template control (NTC). Negative control reactions contained 50 ng of human total RNA. The detection of viral RNA was performed by RT-qLAMP (A) and RT-q(PCR-LAMP) (B–E) assays. The RT-q(PCR-LAMP) assays were carried out with one (B), two (C), four (D), and six (E) PCR cycles before the LAMP isothermal amplification step and provided better sensitivity than the RT-qLAMP assay. The figure shows the amplification reaction time. Time “zero” on the x-axis is the start of the isothermal (LAMP) amplification step. The time required for PCR cycling is highlighted by the gray color at the start of the timeline. All curves are the mean relative fluorescence units RFU of three replicate reactions.