One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

Abstract

Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers.

Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR.

Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR.

Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories.

Keywords: COVID-19; Detection; LAMP Assay; Real time PCR; SARS-CoV-2.

Figure 1.

Locations of primer-binding sequences (Wuhan-Hu-1, complete genome, GenBank Accession No. MN908947.3).

Figure 2.

Analysis of RT-PCR product using 1% agarose gel electrophoresis. A) RT-PCR reaction with total RNA extraction from the patients' respiratory samples. Lane 1: RT-PCR product using primers F3/B3. B) PCR reaction with TA-E plasmid. Lane 1–4: PCR product using primers F3/B3, Lane M: 100 bp DNA leader.

Figure 3.

Analysis of the RT-LAMP reaction product with extracted total RNA (A) and TA-E plasmid (B) as a template. Lane 1: positive control, lane 2: negative control. Top: 1% agarose gel electrophoresis, middle: LAMP results indicated by SYBR green to the visualized under UV light, bottom: view the results with the naked eye.

Figure 4.

A) The specificity of RT-LAMP reaction was determined using Influenza H1N1. Lane 1: total RNA extracted from SARSCoV-2 infected respiratory samples, lane 2: RNA extracted from Influenza H1N1, lane 3: negative control. B) Sensitivity of RT-LAMP reaction using 30 (lane 1) and 15 ng (lane 2) from extracted total RNA of SARS-CoV-2 infected respiratory samples. C) Sensitivity of LAMP reaction using 11.25 ng (lane 1), 1.125 ng (lane 2), and 112 pg (lane 3) of TA-E plasmid. Top: agarose gel electrophoresis, middle: UV analysis, bottom: ocular observation.