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丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri,Mmc)可以引起羊支原体性肺炎(Mycoplasma pneumonia of goats and sheep,MPGS)。现有的方法费时、不能定量,不利于Mmc的准确快速检测。为建立检测Mmc的SYBR Green I qRT-PCR方法,本研究基于Mmc的MLC_1770基因(hypothetical protein gene)序列进行特异性引物设计,用构建的Mmc-1770重组质粒为阳性标准品建立标准曲线,进行了该方法的特异性、灵敏性和重复实验。结果显示:标准曲线相关系数R2为0.999,扩增效率是101.3%;该方法对山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae,Mccp)、绵羊肺炎支原体(Mycoplasma ovipenumoniae,Mo)、巴氏杆菌(Pasteurell amultocida,Pm)、大肠杆菌(Escherichia coli,Ec)、金黄色葡萄球菌(Staphylococcus aureus,SA)及羊传染性脓疱毒(Orf virus,ORFV)均无交叉反应;最低检测限为226拷贝/μL,比常规PCR高100倍;组内变异系数为0.54%~0.79%,组间变异系数为0.70%~0.96%;对临床95份疑似MPGS样品进行检测,阳性率为11.6%(11/95)。实验结果证明该方法特异性强、敏感性高、重复性好,适合于临床样品中Mmc的快速准确检测。
Mycoplasma mycoides subsp. Capri (Mmc) can cause Mycoplasma pneumonia of goats and sheep (MPGS). The existing methods are time-consuming and can not be quantified, which is not conducive to the accurate and rapid detection of Mmc. In order to establish the SYBR Green I qRT-PCR method for detecting Mmc, a specific primer was designed based on Mmc’s MLC_1770 hypothetical protein gene sequence. A standard curve was constructed using the constructed Mmc-1770 recombinant plasmid as a positive standard. The specificity, sensitivity and repeat experiments of this method. The results showed that the correlation coefficient R2 of standard curve was 0.999, and the amplification efficiency was 101.3%. This method could effectively inhibit Mycoplasma capricolum subsp.capripneumoniae (Mccp), Mycoplasma ovipenumoniae (Mo), Pasteurella (Pasteurella multocida, Pm), Escherichia coli (Ec), Staphylococcus aureus (SA) and Orf virus (ORFV). The lowest detection limit was 226 copies / μL, which was 100 times higher than that of the conventional PCR. The coefficient of variation (CV) was 0.54% -0.79% in group and the coefficient of variation was 0.70% -0.96%. 95 samples of suspected MPGS were detected in clinic, the positive rate was 11.6% 95). Experimental results show that the method is specific, sensitive and reproducible, which is suitable for rapid and accurate detection of Mmc in clinical samples.