Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea

文献类型： 外文期刊

第一作者： Wang, Chaonan

作者： Wang, Chaonan;Wang, Fang;Chang, Jitao;Jiang, Zhigang;Han, Yuxin;Wang, Meixi;Yin, Xin;Wang, Chaonan;Jing, Bo;Zhao, Aiyun;Chang, Jitao

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关键词： neonatal calf diarrhea; bovine rotavirus; bovine coronavirus; Escherichia coli k99; real-time PCR

期刊名称：FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY （ 影响因子：5.7； 五年影响因子：5.9 ）

ISSN： 2235-2988

年卷期： 2024 年 14 卷

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收录情况： SCI

摘要： Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect similar to 1 copy/mu L for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/mu L. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research.

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