Development of a novel detection system for microbes from bovine diarrhea by real-time PCR

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  • TSUCHIAKA Shinobu
    The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501–1193, Japan Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • MASUDA Tsuneyuki
    Kurayoshi Livestock Hygiene Service Center, Seidani, Kurayoshi, Tottori 682–0017, Japan
  • SUGIMURA Satoshi
    Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • KOBAYASHI Suguru
    Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • KOMATSU Natsumi
    Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • NAGAI Makoto
    The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501–1193, Japan Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan Laboratory of Epizootiology, Department of Veterinary Medicine Faculty and Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • OMATSU Tsutomu
    The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501–1193, Japan Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • FURUYA Tetsuya
    The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501–1193, Japan Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan Laboratory of Microbiology, Department of Veterinary Medicine Faculty and Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • OBA Mami
    Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • KATAYAMA Yukie
    Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • KANDA Shuhei
    Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • YOKOYAMA Tadashi
    Institute of Agriculture, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan
  • MIZUTANI Tetsuya
    The United Graduate School of Veterinary Sciences, Gifu University, Yanagito, Gifu 501–1193, Japan Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183–8509, Japan

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Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer–probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R2) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16–1.6 TCID50 (PFU/reaction), 1.3–13 CFU/reaction and 10–100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay’s clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

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