Development of droplet digital PCR (ddPCR) for the detection and quantification of Mycoplasma gallisepticum in duck flocks

Abstract

Mycoplasma gallisepticum (MG) is capable of infecting a variety of poultry species, leading to chronic respiratory diseases and posing a significant threat to the poultry industry's development. Although MG infections in chickens have been extensively studied, epidemiological data on ducks remain limited and often underestimated. To address this gap, we developed and validated a ddPCR-based method for the identification and quantification of MG in ducks, using the mgc2 gene sequence. The method's sensitivity, specificity, and reproducibility were evaluated, and clinical samples were tested. The results indicated that the optimal reaction efficiency of the ddPCR was achieved with a primer concentration of 200 nM, a probe concentration of 100 nM, and an annealing temperature of 58.5 °C, resulting in the clearest demarcation between positive and negative droplets. This method has high specificity, with no cross-reactivity observed with other pathogens achieving a minimum detection limit of 10⁰ copies/μL, and increasing tenfold more sensitive than quantitative PCR (qPCR). The coefficient of variation in repeatability tests was below 5 %. Furthermore, analysis of clinical samples revealed that the positive detection rate of ddPCR (53.3 %, 32/60) surpassed that of qPCR (46.7 %, 28/60). This ddPCR method serves as a useful tool for the early diagnosis of MG and assessment of epidemic situation.