TaqMan_MGB探针法实时荧光定量PCR快速检测支原体的研究

目的: 建立特异、敏感、快速检测支原体的TaqMan MGB探针实时荧光定量PCR方法。 方法: 针对支原体16S rRNA基因的保守区设计特异性引物和探针,建立支原体TaqMan MGB探针实时荧光定量PCR方检测方法,验证方法的特异性、敏感性和稳定性。对2008~2010年期间在北京采集的680份小型猪、小鼠、大鼠样本中的支原体进行检测,同时进行分离培养和常规PCR检测。 结果: 建立的TaqMan MGB探针实时荧光定量PCR方法对支原体的检测具有高度的特异性,对空肠弯曲菌、支气管鲍特杆菌、肺炎克雷伯杆菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌、肺炎链球菌、乙型溶血性链球菌均无交叉反应,检测的灵敏度达9.2拷贝。标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.328,TaqMan MGB探针实时荧光定量PCR效率为100%。对680份动物样本进行检测,结果TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出77份支原体阳性样本,但分离培养未能检出支原体阳性样本。结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从动物样本中检出支原体DNA,检测时间仅为2 h。 结论: 本研究建立了一种可靠、快速、灵敏的检测支原体的TaqMan MGB探针实时荧光定量PCR方法,并且成功应用于小型猪、小鼠、大鼠样本中支原体的检测。该技术为动物源性药品和生物制品中支原体的快速检测提供了实用的工具。

Objective: To develop a TaqMan MGB probe-based, sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Mycoplasma. Methods: Primers and probes specific to 16S rRNA gene of Mycoplasma were designed. A TaqMan MGB probe-based, real-time fluorescence quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed. Then, the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Mycoplasma in 680 specimens which collected from miniature pigs, mice and rats during 2008-2010 in Beijing, compared with bacterial isolation and culture method and conventional PCR assay. Results: The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Campylobacter jejuni, Bordetella bronchiseptica, Klebsiella pneumoniae, Pasteurella pneumotropica, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, β-hemolytic streptococcus. The detection limits was 9.2 copies. The correlation coefficient and slope value of standard curve were 0.999 and -3.328, respectively and the efficiency of TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was 100%. The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Mycoplasmas in 680 specimens, a total of 77 specimens were positive for Mycoplasmas. However, Mycoplasmas was not detected in positive specimens by bacteria isolation and culture method. The results showed that TaqMan MGB-based probe real-time fluorescence quantitative PCR for Mycoplasmas was more sensitive than bacteria isolation and culture method, and it could detect Mycoplasmas DNA from animal specimens directly, and detection time was only 2 hours. Conclusion: The established TaqMan MGB probe real-time fluorescence quantitative PCR method is a reliable, fast, and sensitive method and it is successfully used to detect Mycoplasma in specimens of miniature pigs, mice and rats. The technology can provide a useful tool for rapid detection of Mycoplasma in the pharmaceutical and biological products of animal origin.