Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection

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  • 名倉, 由起子
    Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital
  • 呉, 恒寧
    Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital
  • Okamoto, Yuya
    Department of Laboratory Medicine, Osaka Women’s and Children’s Hospital
  • 竹内, 宗之
    Department of Intensive Care Medicine, Osaka Women’s and Children’s Hospital
  • 鈴木, 孝一朗
    The Research Foundation for Microbial Diseases of Osaka University
  • 田村, 嘉孝
    Department of Clinical Laboratory, Osaka Habikino Medical Center
  • 大場, 雄一郎
    Department of General Medicine, Osaka General Medical Center
  • 西海, 史子
    Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital
  • Hatori, Nobuaki
    The Research Foundation for Microbial Diseases of Osaka University
  • 藤原, 伸介
    Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University
  • 保川, 清
    Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University
  • 位田, 忍
    Department of Laboratory Medicine, Osaka Women’s and Children’s Hospital
  • 柳原, 格
    Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital
  • Etsuro Ito
    editor

説明

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 10⁵ copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

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  • PLOS ONE

    PLOS ONE 16 (6), e0252789-, 2021-06

    Public Library of Science (PLoS)

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