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. 2020 Apr 18;21(8):2826.
doi: 10.3390/ijms21082826.

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Renfei Lu  1 , Xiuming Wu  2 , Zhenzhou Wan  3 , Yingxue Li  2 , Xia Jin  4 , Chiyu Zhang  2
Affiliations

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Renfei Lu et al. Int J Mol Sci. .

Abstract

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

Keywords: COVID-19; LAMP; POCT; SARS-CoV-2; pneumonia.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Primer information. (a) Location of the primers in SARS-CoV-2 genome; (b) Sequence comparison among seven human coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, OC43, HKU1, NL63 and 229E).
Figure 2
Figure 2
Specificity of the novel SARS-CoV-2 RT-LAMP assay. The common respiratory viruses used in this assay include: HCoV-HKU-1; HCoV-NL63; HCoV-OC43; HCoV-229E; influenza A, B, and C viruses; parainfluenza virus type 1–3; enterovirus; RSV A and B groups; human rhinovirus; human metapneumovirus; adenovirus; and Bocavirus. SARS-CoV-2 RNA standard was used as positive control (PC). NTC: non-template control.
Figure 3
Figure 3
Sensitivity of the novel SARS-CoV-2 RT-LAMP assay. (a) Real-time monitoring with SYTO9; (b) Visual detection with cresol red. NTC: non-template control.
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