TaqMan荧光定量PCR检测滑液囊支原体方法的建立及其感染鸡血液内载量测定

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

摘要:

【背景】 滑液囊支原体(Mycoplasma synoviae, MS)是禽类的常见病原，严重危害家禽养殖业的发展。MS易与其他病原混合感染，给临床诊断造成困难，因此亟须建立一种快速、准确、灵敏的检测方法对疑似患病禽进行快速诊断，以降低经济损失。【目的】 建立一种检测MS的TaqMan荧光定量PCR方法，测定MS在感染鸡血液内的载量变化，为临床检测MS感染提供技术支持。【方法】 参考GenBank中登录的MS基因保守区域序列设计特异性引物和探针，以温度梯度qPCR法确定最适退火温度，用矩阵法优化其引物和探针加入量，通过提取MS 0801基因组作为模板构建标准曲线，并进行特异性、灵敏度、重复性试验，建立一种检测MS的TaqMan荧光定量PCR方法。使用建立的方法检测滴鼻攻毒和足垫攻毒后MS在感染鸡血液内的载量变化。【结果】 建立的TaqMan荧光定量PCR方法最佳退火温度为55 ℃，引物和探针(10 mol/L)加入量分别为0.8 mL和0.4 mL；扩增曲线具有良好的线性关系，R²>0.99；特异性强，仅对MS有特异性反应，对其他病原均无扩增反应；敏感性高，最低检测限为25 copies/μL；重复性好，批间试验和批内试验变异系数均小于2%。通过TaqMan荧光定量PCR检测MS在感染鸡血液内的动态变化，发现两个攻毒组MS含量的峰值和总体波动无明显差异，但足垫攻毒的MS在血液中的增殖要快于滴鼻攻毒。【结论】 本实验建立了一种能够快速检测MS的TaqMan荧光定量PCR方法，并用于检测MS在感染鸡血液内的动态变化，为临床上检测MS感染提供了技术支撑，为疫苗免疫评价方法提供了新的思路。

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.