Development and validation of a quadruplex real-time quantitative polymerase chain reaction assay for the simultaneous detection of Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Actinobacillus pleuropneumoniae, and Glaesserella parasuis

Background and Aim: Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Actinobacillus pleuropneumoniae, and Glaesserella parasuis are major bacterial pathogens associated with porcine respiratory disease complex (PRDC) and frequently occur as single or mixed infections, causing substantial economic losses to the swine industry. Because these pathogens produce similar clinical signs and pathological lesions, rapid and accurate differential diagnosis is essential for effective disease control. Conventional single-target assays are labor-intensive and less suitable for simultaneous pathogen detection. Therefore, this study aimed to develop and validate a quadruplex real-time quantitative polymerase chain reaction (qPCR) assay for the simultaneous detection of these four important porcine respiratory bacterial pathogens and evaluate its applicability using clinical samples. Materials and Methods: Four pairs of species-specific primers and TaqMan probes targeting the p97 gene of M. hyopneumoniae, p37 of M. hyorhinis, apxIV of A. pleuropneumoniae, and ompA of G. parasuis were designed. The reaction system and amplification conditions were optimized, followed by evaluation of analytical performance, including linearity, sensitivity, specificity, and repeatability. A total of 1,238 clinical samples (821 lung tissues and 417 nasal swabs) collected from pigs in Guangxi Province, China, were tested using the developed assay and reference methods. Diagnostic sensitivity, diagnostic specificity, agreement rates, and pathogen prevalence were determined. Results: The optimized quadruplex assay showed excellent linearity, with correlation coefficients (R²) ≥0.998 for all four targets. The limits of detection were 152.02, 158.53, 161.36, and 140.40 copies/reaction for M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, and G. parasuis, respectively. No cross-reactivity with other common swine pathogens was observed, demonstrating high analytical specificity. The assay also showed excellent repeatability, with intra- and inter-assay coefficients of variation below 2.50%. Among the 1,238 clinical samples, the positivity rates were 31.91% for M. hyopneumoniae, 18.09% for M. hyorhinis, 4.20% for A. pleuropneumoniae, and 26.82% for G. parasuis. Mixed infections were detected in 19.71% of samples, with M. hyorhinis + G. parasuis representing the most frequent co-infection (11.79%). The assay demonstrated diagnostic sensitivities of 96.77%, 95.95%, 95.92%, and 95.87% and diagnostic specificities of 99.40%, 98.92%, 99.58%, and 99.22% for M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, and G. parasuis, respectively, with agreement rates exceeding 98% for all pathogens. Conclusion: The developed quadruplex real-time qPCR assay provides a rapid, sensitive, specific, and reproducible method for the simultaneous detection of four major bacterial pathogens associated with PRDC. Its excellent diagnostic performance and ability to identify mixed infections make it a valuable tool for routine laboratory diagnosis, epidemiological surveillance, and disease control in pig populations, with potential application in large-scale surveillance programs.