Haojie Wang#,Yue Sun#,Jianxing Chen,Wei Wang,Haibo Yu,Caixia Gao,Tongqing An,Yue Wang,Hongyan Chen,Liangquan Zhu,Zhimin Jin,Changqing Yu,Changyou Xia,He Zhang

Front Cell Infect Microbiol.2024 Nov 26:14:1468783.doi: 10.3389/fcimb.2024.1468783. eCollection 2024.

Abstract

Introduction:  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.

Methods: 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.

Results: 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%.

Discussion: 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: Brachyspira hyodysenteriae; Escherichia coli; Lawsonella intracellularis; Quadruplex TaqMan real-time fluorescence quantitative PCR; Salmonella; bacterial gastrointestinal diseases.