SYBR Green Ⅰ RT-qPCR Assay for Mycoplasma ovipneumoniae Detection
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摘要: 根据GenBank公布的绵羊肺炎支原体Y-98株（No.KM435069.1）P80基因序列，利用Beacon Designer 7.9软件设计1对特异性引物，建立了绵羊肺炎支原体的SYBR Green Ⅰ实时荧光定量检测方法。结果显示，该方法可以特异性检测绵羊肺炎支原体，对其他羊常见病原扩增结果无特异性扩增；该方法最低检测限为10 copies·μL-1，组内和组间变异系数均小于2%；采用该方法对96份临床样品进行检测，绵羊肺炎支原体的阳性率为67.7%（65/96），结果显示该方法与TaqMan实时荧光定量PCR方法检测结果一致。以上结果表明本研究建立的绵羊肺炎支原体SYBR Green Ⅰ实时荧光定量PCR方法特异性强、灵敏度高、重复性好，可用于绵羊肺炎支原体的病原学检测和流行病学调查。
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关键词:
- 绵羊肺炎支原体 /
- SYBR Green Ⅰ /
- 荧光定量PCR
Abstract: A SYBR Green Ⅰ RT-qPCR assay was established for detecting the pathogenic Mycoplasma ovipneumoniae in sheep.Using Beacon Designer 7.9 software, a pair of specific primers based on the P80 gene of the M.ovipneumoniae strain Y-98 in Genbank (accession number. No.KM435069.1) was designed for the experiment. The result showed that the new methodology could specifically detect the target pathogen but not any others from sheep or goats. The detection limit was 10 copies·μL-1, and the Ct of intra-and inter-variation all below 2%. The assay method was subsequently applied on 96 clinical samples to yield a positive rate of 67.7% (i.e., 65 out of 96), which was as sensitive as TaqMan RT-PCR. It suggested that the newly established methodology was high on sensitivity, specificity and reproducibility, and applicable for the diagnosis and epidemiologic studies on M.ovipneumoniae.-
Key words:
- Mycoplasma ovipenumon iae /
- SYBR Green Ⅰ /
- RT-qPCR
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图 1 绵羊肺炎支原体阳性质粒扩增
注M为100 bp DNA Ladder；1为绵羊肺炎支原体FJ01-CL质粒DNA；2为阴性对照。
Figure 1. Amplified positive plasmid of M. ovipneumoniae
图 2 绵羊肺炎支原体荧光定量PCR标准曲线
Figure 2. Standard curve of M. ovipneumoniae detected by RT-qPCR
图 3 绵羊肺炎支原体荧光定量PCR熔解曲线
Figure 3. Melting curve of M. ovipneumoniae detected by RT-qPCR
图 4 绵羊肺炎支原体荧光定量PCR特异性试验结果
Figure 4. Specificity test on M. ovipneumoniae detection by RT-qPCR
表 1 绵羊肺炎支原体实时荧光定量PCR的重复性试验结果
Table 1. Repeatability of M.ovipneumoniae detection by RT-qPCR
重组质粒Mo
浓度/
(copies·μL-1)组内重复 组间重复 Ct平均值±
标准差变异系数
/%Ct平均值±
标准差变异系数
/%1×107 13.52±0.19 1.41 13.53±0.18 1.33 1×105 20.19±0.13 0.64 20.13.±0.14 0.69 1×103 26.35±0.47 1.78 26.12±0.43 1.65 
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表 2 SYBR Green Ⅰ荧光定量PCR和TaqMan荧光定量PCR法临床样品检测结果比较
Table 2. M. ovipneumoniae detections on clinical samples using SYBR Green Ⅰ RT-qPCR and TaqMan RT-qPCR
检测方法 样品数 阳性
样品数阴性
样品数阳性率
/%SYBR Green Ⅰ荧光定量PCR 96 65 31 67.7 TaqMan荧光定量PCR 96 65 31 67.7
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