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ORIGINAL RESEARCH article

Front. Cell. Infect. Microbiol.
Sec. Molecular Viral Pathogenesis
Volume 14 - 2024 | doi: 10.3389/fcimb.2024.1540710
This article is part of the Research Topic Detection and Drug Treatment of Emerging Viral Diseases View all 3 articles

Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses

Provisionally accepted
Dequan Yang Dequan Yang 1Li Ma Li Ma 2*Zhongping Yang Zhongping Yang 2*Xianchao Yang Xianchao Yang 1*Jian Wang Jian Wang 1Houbin Ju Houbin Ju 1*Chunguang Lu Chunguang Lu 3*Yonggang Weng Yonggang Weng 3*Heping Zhao Heping Zhao 2*Haixiao Shen Haixiao Shen 1*Xin Li Xin Li 1*Feifei Ge Feifei Ge 1*Xiaoxu Wang Xiaoxu Wang 1*Xiujuan Wu Xiujuan Wu 1*Meng Xiang Meng Xiang 1*Guidan Feng Guidan Feng 1*Congsheng Tang Congsheng Tang 1*Shixin Huang Shixin Huang 1*Hongjin Zhao Hongjin Zhao 1*
  • 1 Shanghai Animal Disease Prevention and Control Center, Shanghai, China
  • 2 Hunan Guanmu Biotech Co., Ltd, Changsha, China
  • 3 Jinshan District Animal Disease Control Center, Shanghai, China

The final, formatted version of the article will be published soon.

    Viral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). This novel method demonstrated high sensitivity and specificity, achieving a detection limit of 24 copies/μL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R² > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with conventional reverse-transcription PCR (RT-PCR), with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region. In summary, the established mRT-qPCR assay serves as a critical tool for the rapid clinical detection of viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

    Keywords: Calf diarrhea, multiplex real-time quantitative PCR (mRT-qPCR), Clinical detection, Method, epidemiological surveillance

    Received: 06 Dec 2024; Accepted: 23 Dec 2024.

    Copyright: © 2024 Yang, Ma, Yang, Yang, Wang, Ju, Lu, Weng, Zhao, Shen, Li, Ge, Wang, Wu, Xiang, Feng, Tang, Huang and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Li Ma, Hunan Guanmu Biotech Co., Ltd, Changsha, China
    Zhongping Yang, Hunan Guanmu Biotech Co., Ltd, Changsha, China
    Xianchao Yang, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Houbin Ju, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Chunguang Lu, Jinshan District Animal Disease Control Center, Shanghai, China
    Yonggang Weng, Jinshan District Animal Disease Control Center, Shanghai, China
    Heping Zhao, Hunan Guanmu Biotech Co., Ltd, Changsha, China
    Haixiao Shen, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Xin Li, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Feifei Ge, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Xiaoxu Wang, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Xiujuan Wu, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Meng Xiang, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Guidan Feng, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Congsheng Tang, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Shixin Huang, Shanghai Animal Disease Prevention and Control Center, Shanghai, China
    Hongjin Zhao, Shanghai Animal Disease Prevention and Control Center, Shanghai, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.