Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening

Authors

  • Kazuya Takizawa,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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    • These authors contributed equally.
  • Tatsuo Nakashima,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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    • These authors contributed equally.
  • Takuo Mizukami,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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    • These authors contributed equally.
  • Madoka Kuramitsu,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Daiji Endoh,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Shigeto Kawauchi,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Kohsuke Sasaki,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Haruka Momose,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Yoshiharu Kiba,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Tetsuya Mizutani,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Rika A. Furuta,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Kazunari Yamaguchi,

    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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  • Isao Hamaguchi

    Corresponding author
    1. Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan
    2. Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
    3. Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan
    4. Osaka Red Cross Blood Center, Osaka, Japan
    5. Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
    • Address reprint requests to: Isao Hamaguchi, Department of Safety Research on Blood and Biologics, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan; e-mail: 130hama@nih.go.jp.

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  • This study was supported by a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan.

Abstract

Background

The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level.

Study Design and Methods

We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes.

Results

We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses.

Conclusion

We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

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