Enhancing Mycoplasma synoviae control: A novel quantitative real-time PCR approach to differentiate MS-H vaccine strain and field strains.

Mycoplasma synoviae (MS) is a prevalent pathogen in poultry, causing substantial economic losses. Vaccination is a crucial strategy for controlling MS infections. In China, a temperature-sensitive live vaccine, Vaxsafe®MS (MS-H strain), was introduced to address the rising prevalence of MS since 2017. Accurate differentiation between the MS-H vaccine strain and field strains is essential for the effective application of the vaccine and the eradication of MS in poultry. In this study, a duplex TaqMan real-time quantitative PCR (qPCR) method was first developed based on a single nucleotide polymorphism (SNP) at position 367 of the obg gene. However, some reisolated strains of the MS-H vaccine strain may exhibit reverse mutations at this site, potentially leading to false-positive results field strains detection. To address this issue, a comprehensive comparison of 24 MS genomes was conducted, identifying 18 SNPs unique to the MS-H lineage. Subsequently, an (A/G) SNP at position 131 of the MSH_02330 gene was selected through sequencing. Based on this SNP, a novel duplex qPCR method was established, characterized by high sensitivity, specificity, and repeatability. This method eliminates the influence of obg revertant strains, enabling accurate differentiation of the MS-H vaccine strain from MS field strains. It can be applied to both pure cultures and clinical samples, serving as a valuable tool for evaluating the immunogenicity of the MS-H vaccine and advancing the prevention and control of MS infections.