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ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Veterinary and Zoonotic Infection
Volume 14 - 2024 |
doi: 10.3389/fcimb.2024.1468783
This article is part of the Research Topic Unveiling Host-Pathogen Interactions: Insights into Animal Cellular Immunity and Novel Diagnostics View all 20 articles
Development and application of a quadruplex TaqMan real-time fluorescence quantitative PCR assay for four porcine digestive pathogens
Provisionally accepted
Haojie Wang Haojie Wang
1
Yue Sun
2
Jianxing Chen
1*
Wei Wang
1*
Haibo Yu
1*
Caixia Gao
1
Tongqing An
1*
Yue Wang
1*
Hongyan Chen
1*
Liangquan Zhu
3*
Zhimin Jin
2*
Changqing Yu
4*
Changyou Xia
1*
He Zhang
1*- 1 Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
- 2 Mudanjiang Normal University, Mudanjiang, China
- 3 China Institute of Veterinary Drug Control, Beijing, Beijing Municipality, China
- 4 Yibin Vocational and Technical College, Yibin, Sichuan Province, China
Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections. In this study, we designed primers and probes targeting the invA gene of Salmonella, the 23S rRNA gene of Escherichia coli, the aspA gene of Lawsonella intracellularis, and the nox gene of Brachyspira hyodysenteriae. We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens. This assay demonstrated high sensitivity, with detection limits of 100 copies/μL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-nox, and 10 copies/μL for pEASY-invA. The standard curves exhibited excellent linearity (R² values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), Pasteurella multocida, Clostridium perfringens, Gracilaria parapsilosis, porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter-and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected Salmonella in 23.95%, Escherichia coli in 26.24%, Lawsonella intracellularis in 33.84%, and Brachyspira hyodysenteriae in 22.43%. This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae in pigs.
Keywords: Bacterial gastrointestinal diseases, Salmonella, Escherichia coli, Lawsonella intracellularis, Brachyspira hyodysenteriae, Quadruplex TaqMan
Received: 22 Jul 2024; Accepted: 29 Oct 2024.
Copyright: © 2024 Haojie Wang, Sun, Chen, Wang, Yu, Gao, An, Wang, Chen, Zhu, Jin, Yu, Xia and Zhang. 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:
Jianxing Chen, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Wei Wang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Haibo Yu, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Tongqing An, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Yue Wang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Hongyan Chen, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Liangquan Zhu, China Institute of Veterinary Drug Control, Beijing, Beijing Municipality, China
Zhimin Jin, Mudanjiang Normal University, Mudanjiang, China
Changqing Yu, Yibin Vocational and Technical College, Yibin, Sichuan Province, China
Changyou Xia, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
He Zhang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
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