Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification

Abstract

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

Fig 1. RT-LAMP detection of SARS-CoV-2.

(A) SARS-CoV-2 RT-LAMP amplification of SARS-CoV-2 PCR standard (SARS-CoV-2; IDT custom oligo) but not no template control (NTC; negative control) as visualized by addition of SYBR Green I (SYBR) by eye (upper panel), fluorescence (middle panel), or gel electrophoresis (bottom panel). All primers (ALL) are required for effective RT-LAMP reaction. Reactions without FIP and BIP (-FIP/BIP) or FL and BL (-FL/BL) primers resulted in a negative RT-LAMP reaction.

Fig 2. SARS-CoV-2 RT-LAMP sensitivity for SARS-CoV-2.

Sensitivity assessment of SARS-CoV-2 RT-LAMP using serial dilutions of SARS-CoV-2 PCR Standard from 10.0 ng/reaction to 0.06 fg/reaction as visualized by addition of SYBR Green I by eye (upper panel), fluorescence (middle panel), or gel electrophoresis (bottom panel). NTC: No template control (negative control).

Fig 3. SARS-CoV-2 RT-LAMP specificity for SARS-CoV-2 in simulated patient samples.

Specificity assessment of SARS-CoV-2 RT-LAMP in control samples (control) or samples spiked with SARS-CoV-2, MERS, BtCoV, MHV PCR standards (IDT custom oligos) as visualized by the addition of SYBR Green I by eye (upper panel), fluorescence (middle panel), or gel electrophoresis (bottom panel). Types of human samples tested included serum, urine, saliva, nasopharyngeal swabs (nasal swab), or oropharyngeal swabs (oral swab). NTC: No template control (negative control). 3–5 different patient samples were tested for each condition with one representative patient being shown.

Fig 4. Detection of SARS-CoV-2 with RT-LAMP in patient nasopharyngeal swab samples with RNA isolation.

RNA isolated from patients with COVID-19 symptoms with (A) or without (B) SARS-CoV-2 detection by qRT-PCR were tested for SARS-CoV-2 by RT-LAMP. SARS-CoV-2 detection by RT-LAMP was visualized by the addition of SYBR Green I by eye (upper panels), fluorescence (middle panels), or gel electrophoresis (bottom panels).

Fig 5. Detection of SARS-CoV-2 with RT-LAMP in patient nasopharyngeal swab samples without RNA isolation.

Nasopharyngeal swabs in viral transport media from patients with COVID-19 symptoms with (A) or without (C) SARS-CoV-2 detection by qRT-PCR were tested for SARS-CoV-2 by RT-LAMP. SARS-CoV-2 detection by RT-LAMP was visualized by the addition of SYBR Green I by eye (upper panels), fluorescence (middle panels), or gel electrophoresis (bottom panels). B) Average Ct values from qRT-PCR performed on isolated RNA from the same samples in panel A are listed for target SARS-CoV-2 genes N1 and N2.