Development and Application of a Real-time Fluorescence Quantitative PCR for Detection of Orf Virus
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摘要: 根据GenBank公布的羊传染性脓疱病毒VIR基因序列（No.KF666563.1），利用Beacon Designer 7.9软件设计1对特异性引物，建立羊传染性脓疱病毒VIR基因SYBR Green I实时荧光定量PCR检测方法。结果显示，该方法可以特异性检测羊传染性脓疱病毒，对羊常见病原无特异性扩增；最低检测限为100 copies·μL-1；组内和组间变异系数均小于2%；应用该方法对39份临床样品进行检测，阳性率为94.9%（37/39）。以上结果表明该方法特异性强、灵敏度高、重复性好，适合于羊传染性脓疱的病原学检测和流行病学调查。
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关键词:
- 羊传染性脓疱病毒 /
- SYBR Green I /
- 实时荧光定量PCR
Abstract: The study established a SYBR Green I real-time fluorescence quantitative PCR assay for detecting orf virus. Using Beacon Designer 7.9 software to design a pair of specific primers based on the VIR gene of the orf virus in the Genbank (accession number.KF666563.1). The result showed that it would specifically detect orf virus, but wouldn't detect other sheep and goat bacteria and viruses. The detection limit of the method was 100 copies·μL-1 and the detected Ct values of intra-and inter-variation were all less than 2%. Using the assay to detect 39 clinical samples, the positive rate was 94.9% (37/39). The above results indicated that the assay had a high specificity, sensitivity and repeatability, which could be used to detect orf virus and conduct epidemiological investigation.-
Key words:
- orf virus /
- SYBR Green I /
- real-time fluorescence quantitative PCR
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图 1 目的片段的PCR扩增
注：M为DNA标准DL 1 000；1为羊传染性脓疱病毒DNA；2为阴性对照。
Figure 1. PCR results of target DNA fragment
图 2 阳性质粒荧光PCR扩增
Figure 2. The fluorescence quantitative PCR amplification of positive plasmid
图 5 荧光定量PCR特异性试验
注:1为羊传染性脓疱病毒；2为绵羊肺炎支原体；3为丝状支原体山羊亚种；4为大肠杆菌；5为多杀性巴氏杆菌；6为金黄色葡萄球菌；7为溶血性曼氏杆菌；8为牛支原体；9为莱氏无胆甾原体；10为双蒸水。
Figure 5. Specificity test for fluorescence quantitative PCR
表 1 羊传染性脓疱病毒实时荧光定量PCR的重复性实验结果
Table 1. Repeatability test results of the real time fluorescence quantitative PCR for ORFV
重组质粒ORFV浓度/(copies·μL-1) 组内重复Ct平均值±标准差 变异系数/% 组间重复Ct平均值±标准差 变异系数/% 1×107 15.19±0.27 1.78 15.23±0.18 1.18 1×105 22.22±0.24 1.08 22.19.±0.26 1.17 1×103 27.95±0.33 1.18 27.93±0.32 1.15 
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表 2 荧光定量PCR和常规PCR检测临床样品结果比较
Table 2. Comparison of detecting results of clinical samples by using fluorescence quantitative PCR and conventional PCR
检测方法 样品数 阳性样品数 阴性样品数 阳性率/% 荧光定量PCR 39 37 2 94.9 常规PCR 39 35 4 89.7
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