Strain-specific detection of Mycoplasma gallisepticum: A duplex qPCR assay for vaccine efficacy and infection surveillance.

Mycoplasma gallisepticum (MG) is a major pathogen of poultry that causes substantial economic losses to the global poultry industry. Vaccination with live attenuated vaccines is currently a core strategy for controlling MG infection in chickens. In China, the officially approved MG vaccine strains for poultry use include F-36, TS-11, and 6/85. Following vaccination, effective methods are required to evaluate vaccine strain colonization and differentiate wild-type from vaccine strain infections. To address this unmet need, we developed a set of rapid, specific duplex quantitative PCR (qPCR) methods based on MGB probes. This method can discriminate between wild-type MG and all three currently used major vaccine strains, and users can select the corresponding specific duplex combination according to the vaccine strain adopted in field production. First, we compared whole-genome sequences of wild-type MG and vaccine strains, and successfully identified strain-specific polymorphic sites. Next, we designed and optimized specific primers and MGB probes targeting these polymorphic sites. Using the developed primer-probe sets, we established three independent duplex qPCR assays, each of which enables simultaneous detection of one specific vaccine strain and wild-type MG. Validation experiments confirmed that all three assays have high specificity, sensitivity, and reproducibility. When applied to DNA extracted from clinical chicken choanal cleft swab samples, this method successfully differentiated between wild-type MG infection and vaccine strain exposure. In conclusion, this novel assay provides a valuable technical tool for MG surveillance, MG eradication programs, and vaccine efficacy monitoring.