猪肺炎支原体实时荧光定量PCR检测方法建立及应用

Establishment and Application of Real-time Fluorescence Quantitative PCR Method For Detection of Mycoplasma hyopneumoniae

猪肺炎支原体(Mycoplasma hyopneumoniae)是一种严重危害养猪业的重要传染性病原,以猪肺炎支原体编码乳酸脱氢酶的P 36基因保守序列为基础,设计一套特异性引物和探针,并通过优化筛选反应条件,建立了一种Mhp的实时荧光定量RT-PCR检测方法。结果表明,最适引物浓度为10μmol/L,探针浓度为5μmol/L,以浓度为1×10^(2)~1×10^(6)copies/μL的标准品构建标准曲线相关系数为0.998,扩增效率可达96%。该方法能特异地检测猪肺炎支原体,与其他动物支原体及猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病病毒等病原无交叉反应;灵敏度是常规PCR的10倍,可达到10拷贝;该方法重复性较好,组内和组间变异系数均小于2%。在对30份临床样本的检测中,建立的实时荧光定量PCR检出率为63%(19/30),而常规PCR的检出率仅有30%(9/30)。结果表明,成功建立了猪肺炎支原体实时荧光定量PCR检测方法,可用于猪肺炎支原体的病原学检测和流行病学调查。

Mycoplasma hyopneumoniae(Mhp)is a significant infectious pathogen that poses a substantial threat to the pig industry.In this study,a set of specific primers and probes were designed based on the conserved sequence of the lactate dehydrogenase gene P36 of Mhp.The real-time fluorescence quantitative RT-PCR method for Mhp detection was established by optimizing the screening reaction conditions.The results indicated that the optimal primer concentration was 10μmol/L,the probe concentration was 5μmol/L,and the correlation coefficient of the standard curve was 0.998,demonstrating a concentration range of 1×10^(2)to 1×10^(6)copies/μL.The method specifically detects Mhp without any cross-reaction with other animal Mycoplasma,porcine reproductive and respiratory syndrome virus,porcine circovirus type 2,classical swine fever virus,pseudorabies virus,and other pathogens.It exhibits excellent repeatability,with a coefficient of variation within and between groups of less than 2%.The sensitivity is ten fold higher than that of conventional PCR and capable of detecting as low as ten copies.Among the thirty clinical samples tested,the Taq Man fluorescence quantitative PCR established in this study exhibited a detection rate of 63%(19/30),whereas conventional PCR only achieved 30%(9/30).In summary,this study successfully developed a Taq Man probe-based real-time fluorescence quantitative PCR assay for detection and epidemiological investigation of Mhp.