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Simultaneous quantitative detection of 12 pathogens using high-resolution CE-SSCP

Authors

  • Gi Won Shin,

    1. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea
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    • These authors are co-first authors.

  • Hee Sung Hwang,

    1. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea
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    • These authors are co-first authors.

  • Mi-Hwa Oh,

    1. National Institute of Animal Science, Rural Development Administration, Suwon, Korea
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  • Junsang Doh,

    1. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea
    2. Department of Mechanical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea
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  • Gyoo Yeol Jung

    Corresponding author
    1. Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea
    2. School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea
    • Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Korea Fax: +82-54-279-5528
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Abstract

Several methods based on screening for a 16S ribosomal RNA gene marker have been developed for rapid and sensitive detection of pathogenic microorganisms. One such method, CE-based SSCP (CE-SSCP), has enormous potential because the technique can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S ribosomal RNA gene-specific markers unless combined with additional modification steps. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this study, we developed a method based on a high-resolution CE-SSCP system using a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer that is capable of simultaneous and quantitative detection of 12 clinically important pathogens. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing easy identification of pathogens. A series of experiments using different amounts of genomic pathogen DNA showed that the method had a limit of detection of 0.31–1.56 pg and a dynamic range of approximately 102. These results indicate that high-resolution CE-SSCP systems have considerable potential in the clinical diagnosis of bacteria-induced diseases.

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