A multiplex TaqMan real-time PCR assay for differential identification of wild-type and vaccine strains of Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) is a major avian respiratory pathogen responsible for chronic respiratory disease and substantial economic losses in the poultry industry worldwide. Live attenuated vaccines, including strains F, ts-11 , and 6/85 , are widely used for MG control; however, current diagnostic methods are unable to reliably differentiate wild-type MG infections from vaccine strain persistence or replication, complicating surveillance and control programs in vaccinated flocks. In this study, a rapid and discriminatory multiplex TaqMan real-time quantitative PCR (qPCR) assay was developed to simultaneously detect MG and differentiate wild-type strains from the three major commercial live vaccine strains ( F, ts-11 , and 6/85 ) in a single reaction. Strain- and species-specific target regions were identified through comparative genomic analysis of 19 MG genomes, and corresponding primers and probes were designed and optimized. Under optimal conditions, the assay exhibited excellent linearity (R 2 = 0.995-0.998), amplification efficiencies ranging from 85.16% to 96.40%, and a limit of detection of 1 × 10 2 copies/μL for all targets, demonstrating a 10-fold higher sensitivity than conventional PCR. No cross-reactivity was observed with other common avian pathogens, including NDV, AIV, IBV, FAdV, ILTV, and MS. Application of the assay to clinical poultry samples demonstrated its ability to reliably distinguish vaccine strains from wild-type MG. Using this method, the prevalence of wild-type MG was investigated in 2,564 poultry samples collected in the Guangxi region. The results showed that vaccination was associated with inhibition of wild-type MG infection within flocks, whereas unvaccinated flocks exhibited a wild-type MG infection rate of 58.5% (416/711). In conclusion, this multiplex qPCR assay provides a sensitive and specific tool for differentiating wild-type MG from vaccine strains and may support routine diagnosis and improved control of MG in poultry production systems.