猪肺炎支原体SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

Establishment and Application of SYBR Green I Real-time Fluorescence Quantitative PCR Assay for Mycoplasma hyopneumoniae

为建立一种快速高效的猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)检测方法,根据NCBI公布的Mhp保守序列设计4对特异性引物,依据4对引物的荧光定量PCR检测结果及拟合曲线进行相关性分析,筛选出最佳引物(SX4),建立了Mhp SYBR Green Ⅰ实时荧光定量检测方法。该方法可特异性检测Mhp,对猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、副猪嗜血杆菌4型及链球菌2型等病原均无特异性扩增;最佳引物SX4检测的灵敏度可达4.9×10^(1) copies/μL,组内和组间变异系数均小于2%。该方法可稳定检测出工艺生产抗原和市售疫苗中的Mhp菌量。试验表明,本研究成功建立了Mhp SYBR Green Ⅰ实时荧光定量检测方法,其特异性强、灵敏度高、重复性好,可用于Mhp疫苗抗原生产和临床疫苗中的Mhp菌量检测。

In order to establish a rapid and efficient method to detect Mycoplasma hyopneumoniae(Mhp),4 pairs of specific primers were designed based on the conserved sequence of Mhp published by NCBI,and the optimal primer(SX4)was screened after correlation analysis based on the test results of the primers by fluorescence quantitative PCR and fitting curves to establish a Mhp SYBR Green Ⅰ real-time fluorescence quantitative PCR assay.The method could be used to specifically detect Mhp,without specific amplification to porcine circovirus type 2,porcine parvovirus,pseudorabies virus,Haemophilus parasuis serotype 4 and Streptococcus suis serotype 2.The sensitivity of SX4 was up to 4.9×10^(1) copies/μL,and inter/intra-group variable coefficient was less than 2%.The method could be used to detect the amount of Mhp from produced antigen and commercial vaccines.In conclusion,the Mhp SYBR Green I real-time fluorescence quantitative PCR assay was successfully established,and could be used to detect the amount of Mhp from produced antigen and clinical vaccines with the advantages of strong specificity,high sensitivity and good repeatability.