Using a novel gene site to develop a duplex real-time TaqMan MGB probe PCR method for the SNP detection and differentiation between the MS-H live vaccine strain and wild-type Mycoplasma synoviae strains

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Mycoplasma synoviae (MS) is a globally prevalent avian pathogen responsible for airsacculitis and synovitis. The temperature-sensitive (ts)+ vaccine strain MS-H, a live attenuated variant, is the most effective and widely used vaccine for controlling infections in the poultry industry. Consequently, accurate detection is essential for a strategy known as differentiating infected from vaccinated animals (DIVA). In this study, we developed a duplex real-time TaqMan minor groove binder (MGB) probe PCR (The DRTM-probe PCR) method to differentiate the MS-H live vaccine strain from wild-type strains by targeting a single nucleotide polymorphism (SNP) in the ktrB gene. This gene overcomes the restoration of the genotype of wild-type 86079/7NS in specific regions. With a detection limit of 6.25 copies/μL, the DRTM-probes PCR method demonstrates a good specificity in distinguishing in one hour. For simulated clinical samples, the method achieved over 95 % sequence identity with reference fragments, confirming its accuracy. The established DRTM-probe PCR method offers a specific, rapid, and reliable approach for SNP detection with significant application potential.