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

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

JOURNAL=Frontiers in Veterinary Science

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2024.1450066

DOI=10.3389/fvets.2024.1450066

ISSN=2297-1769

ABSTRACT=<sec id="sec1"><title>Introduction</title><p>PEDV, <italic>Brachyspira hyodysenteriae,</italic> and <italic>Lawsonia intracellularis</italic>, 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.</p></sec><sec id="sec2"><title>Methods</title><p>Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of <italic>B. hyodysenteriae</italic>, and the 16S rDNA gene of <italic>L. intracellularis</italic>, 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.</p></sec><sec id="sec3"><title>Results</title><p>The 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 <italic>B. hyodysenteriae</italic> and <italic>L. intracellularis</italic>, 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, <italic>B. hyodysenteriae</italic> and <italic>L. intracellularis</italic>. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for <italic>B. hyodysenteriae</italic>, and 98.6% for <italic>L. intracellularis</italic>. The co-infection rates were 16.85% for PEDV + <italic>B. hyodysenteriae</italic>, 31.46% for PEDV + <italic>L. intracellularis</italic>, 57.86% for <italic>B. hyodysenteriae</italic> + <italic>L. intracellularis</italic>, and 16.85% for PEDV + <italic>B. hyodysenteriae</italic> + <italic>L. intracellularis</italic>, respectively.</p></sec><sec id="sec4"><title>Discussion</title><p>The new multiplex real-time PCR method can simultaneously differentiate PEDV, <italic>B. hyodysenteriae</italic> and <italic>L. intracellularis</italic>, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.</p></sec>