Frontiers in veterinary science · 2026
Development and validation of a multiplex TaqMan real-time quantitative PCR assay for the simultaneous detection of six major bacterial respiratory pathogens in goats.
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Fu Mingzhe, Liu Yi, Zhang Zeyang, Li Weimin, Zhang Qi, Xu Xingang
Аннотация
BACKGROUND: Respiratory disease caused by bacterial and mycoplasmal pathogens is an important threat to goat health, and mixed infections complicate etiological diagnosis. Rapid multiplex detection is therefore valuable for clinical investigation and surveillance.
METHODS: Two triplex TaqMan real-time quantitative PCR (qPCR) panels were established for the simultaneous detection of six major caprine respiratory pathogens. Panel I targeted Mycoplasma ovipneumoniae (Mo), Mycoplasma mycoides subsp. capri (Mmc), and Mycoplasma capricolum subsp. capripneumoniae (Mccp), whereas Panel II targeted Pasteurella multocida (Pm), Mannheimia haemolytica (Mh), and Streptococcus equi subsp. zooepidemicus (Sz). Analytical specificity was assessed using target plasmids and nucleic acids from target and non-target pathogens, whereas analytical sensitivity and reproducibility were evaluated using recombinant plasmid standards. Clinical applicability was assessed separately using 200 field-collected goat nasal swab samples.
RESULTS: No amplification was observed for the tested non-target organisms. The limits of detection were 1.73 × 102 copies/μL for Mo, 2.01 × 103 copies/μL for Mmc, 3.63 × 102 copies/μL for Mccp, 1.03 × 102 copies/μL for Pm, 1.59 × 102 copies/μL for Mh, and 5.88 × 102 copies/μL for Sz. Intra-assay CVs ranged from 0.55 to 2.76%, and inter-assay CVs ranged from 0.38 to 3.10%. Multiplex qPCR detected at least one target in 117 of 200 clinical samples (58.5%), including 47 samples with two or more targets (23.5%).
CONCLUSION: The multiplex qPCR assay developed in this study provides a rapid and specific tool for the simultaneous detection of six major respiratory pathogens in goats, with potential utility for clinical diagnosis and epidemiological surveillance.
METHODS: Two triplex TaqMan real-time quantitative PCR (qPCR) panels were established for the simultaneous detection of six major caprine respiratory pathogens. Panel I targeted Mycoplasma ovipneumoniae (Mo), Mycoplasma mycoides subsp. capri (Mmc), and Mycoplasma capricolum subsp. capripneumoniae (Mccp), whereas Panel II targeted Pasteurella multocida (Pm), Mannheimia haemolytica (Mh), and Streptococcus equi subsp. zooepidemicus (Sz). Analytical specificity was assessed using target plasmids and nucleic acids from target and non-target pathogens, whereas analytical sensitivity and reproducibility were evaluated using recombinant plasmid standards. Clinical applicability was assessed separately using 200 field-collected goat nasal swab samples.
RESULTS: No amplification was observed for the tested non-target organisms. The limits of detection were 1.73 × 102 copies/μL for Mo, 2.01 × 103 copies/μL for Mmc, 3.63 × 102 copies/μL for Mccp, 1.03 × 102 copies/μL for Pm, 1.59 × 102 copies/μL for Mh, and 5.88 × 102 copies/μL for Sz. Intra-assay CVs ranged from 0.55 to 2.76%, and inter-assay CVs ranged from 0.38 to 3.10%. Multiplex qPCR detected at least one target in 117 of 200 clinical samples (58.5%), including 47 samples with two or more targets (23.5%).
CONCLUSION: The multiplex qPCR assay developed in this study provides a rapid and specific tool for the simultaneous detection of six major respiratory pathogens in goats, with potential utility for clinical diagnosis and epidemiological surveillance.