Frontiers in Veterinary Science (Aug 2024)

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis

  • Jing Ren,
  • Jing Ren,
  • Fujun Li,
  • Xue Yu,
  • Xue Yu,
  • Yang Li,
  • Meng Li,
  • Meng Li,
  • Yujie Sha,
  • Yujie Sha,
  • Xiaowen Li,
  • Xiaowen Li

DOI
https://doi.org/10.3389/fvets.2024.1450066
Journal volume & issue
Vol. 11

Abstract

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IntroductionPEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens.MethodsBased on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method.ResultsThe approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for B. hyodysenteriae and L. intracellularis, and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively.DiscussionThe new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

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