In this study, reference materials (RMs) were developed to enable quantitative comparability for six major swine bacterial pathogens, including Actinobacillus pleuropneumoniae, Glaesserella parasuis, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, and Salmonella enterica subsp. enterica serovar Typhimurium. These pathogens impose significant health and economic burdens on global swine production through severe respiratory diseases, systemic infections, polyserositis, arthritis, and zoonotic risks, resulting in increased mortality, reduced performance, and substantial treatment and vaccination costs. The optimized primer-probe assay targeted conserved genomic regions for each species to ensure broad detection capabilities across relevant strains using droplet digital PCR (ddPCR). RMs were prepared by combining bacterial genomic DNA with a swine tissue matrix mixture, followed by a comprehensive assessment of homogeneity and stability. The ddPCR-based approach demonstrated high reproducibility and sensitivity for homogeneity and stability assessment of RMs, providing absolute quantification without standard curves. Between-bottle homogeneity showed variations of 6.4-22.9%, representing the relative variation among aliquots observed during homogeneity assessment, while stability assessments confirmed reliability during storage at various temperatures and through multiple freeze-thaw cycles. Each RM was assigned a reference value with well-characterized measurement uncertainty. These RMs support quantitative comparability of analytical results under consistent analytical conditions and provide reliable reference values for evaluating diagnostic assays and standardizing molecular testing. However, further comparative evaluations across different analytical platforms, laboratories, and workflows are required to extend their applicability to diverse diagnostic environments.

To Cite This Article: Park J, Park C, Jeon GT, Ku BK, Byun JW, Hwang MH, Park M and Kim S, 2026. Development of reference materials enabling quantitative comparability for six swine bacterial pathogens. Pak Vet J, 46(9): 2220-2229. http://dx.doi.org/10.29261/pakvetj/2026.210