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

• Published in: Emerging microbes & infections 2020-01, Vol.9 (1), p.998-1007
• Main Authors: Baek, Yun Hee, Um, Jihye, Antigua, Khristine Joy C., Park, Ji-Hyun, Kim, Yeonjae, Oh, Sol, Kim, Young-il, Choi, Won-Suk, Kim, Seong Gyu, Jeong, Ju Hwan, Chin, Bum Sik, Nicolas, Halcyon Dawn G., Ahn, Ji-Young, Shin, Kyeong Seob, Choi, Young Ki, Park, Jun-Sun, Song, Min-Suk
• Format: Article
• Language: English
• Subjects: Betacoronavirus - genetics; Betacoronavirus - isolation & purification; colorimetric detection; Coronavirus Infections - diagnosis; Coronavirus Infections - virology; Coronavirus Nucleocapsid Proteins; Coronaviruses; COVID-19; Disease control; Humans; Laboratories; Limit of Detection; Middle East respiratory syndrome; molecular diagnosis; novel coronavirus; 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; reverse transcription-loop-mediated isothermal amplification; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; Time Factors
• ISSN: 2222-1751; 2222-1751
• DOI: 10.1080/22221751.2020.1756698

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 10 2 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.