Establishment of TaqMan fluorescence quantitative PCR method for detecting Mycoplasma synovium and determination of blood load in infected chickens

Abstract:

[Background] Mycoplasma synoviae (MS) is a common pathogen in poultry, posing a serious threat to the development of the poultry industry. The co-infection of MS with other pathogens makes it difficult to diagnose based on clinical symptoms. Therefore, it is urgent to establish a rapid, accurate, and sensitive method for rapid diagnosis of suspected sick birds to reduce economic losses. [Objective] To establish a TaqMan fluorescence quantitative PCR method for determining the load of MS in the blood of infected chicken, and to provide technical support for the clinical detection of MS infection. [Methods] A pair of specific primers and a probe were designed according to the conserved sequence of MS gene in GenBank. The optimal annealing temperature was determined by temperature gradient qPCR, and the amounts of primers and the probe were optimized by the matrix method. The standard curve was established with the genome of MS 0801 as a template, and the specificity, sensitivity, and repeatability of the established method were evaluated. Furthermore, the established method was used to measure the MS load in the blood of chicken samples infected via nasal drip or footpad challenge. [Results] The optimal annealing temperature of the established method was 55 ℃, and the amounts of primers and the probe (10 mol/L) were 0.8 mL and 0.4 mL, respectively. The amplification curve had a good linear relationship, with R²>0.99. The method had strong specificity since it had specific reaction only to MS and no reaction to other pathogens. In addition, the method had high sensitivity, with the lower limit of detection being 25 copies/μL blood. The coefficients of variation of inter-batch and intra-batch experiments were less than 2%, which indicated that the method had high repeatability. There was no significant difference in the peak or overall fluctuations of MS load in the chicken samples infected via nasal drip or footpad challenge, while the proliferation of MS in the blood of footpad challenge was faster than that of nasal drip challenge. [Conclusion] A TaqMan fluorescence quantitative PCR method for rapid detection of MS was established and used to detect the dynamic changes of MS in the blood of infected chicken. This method provided technical support for clinical detection of MS infection and new ideas for evaluation of vaccination performance.

Key words:Mycoplasma synoviae;TaqMan fluorescence quantitative PCR;challenge model;proliferation changes