Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2

Emerg Microbes Infect. 2020 Dec;9(1):998-1007. doi: 10.1080/22221751.2020.1756698.

Abstract

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.

Keywords: COVID-19; SARS-CoV-2; colorimetric detection; molecular diagnosis; novel coronavirus; reverse transcription-loop-mediated isothermal amplification.

MeSH terms

  • Betacoronavirus / genetics
  • Betacoronavirus / isolation & purification
  • COVID-19
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Coronavirus Nucleocapsid Proteins
  • Humans
  • Limit of Detection
  • Nucleic Acid Amplification Techniques / economics
  • Nucleic Acid Amplification Techniques / methods*
  • Nucleic Acid Amplification Techniques / standards
  • Nucleocapsid Proteins / genetics
  • Pandemics
  • Phosphoproteins
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • SARS-CoV-2
  • Time Factors

Substances

  • Coronavirus Nucleocapsid Proteins
  • Nucleocapsid Proteins
  • Phosphoproteins
  • nucleocapsid phosphoprotein, SARS-CoV-2

Grants and funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) [grant numbers NRF-2018R1A2B2005086 to M-.S.S., NRF-2018R1A1A3A04077961 to Y.H.B and NRF-2018M3A9H4055769 to Y.H.B].