ORFV和Mo双重PCR检测方法的建立及应用

发布时间：2018-11-20 11:06

【摘要】：羊传染性脓疱病毒(Orf virus,ORFV)是羊传染性脓疱病(Contagious ecthyma,CE)的病原,绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)是羊支原体性肺炎(Mycoplasma pneumonia of goats and sheep,MPGS)的主要病原,近年来此两种病原混合感染日益增多,目前采用单一PCR方法检测这两种病原,临床上迫切需要能同时快速检测这两种病原的方法。为了建立能够直接从样品中同时检测ORFV核酸和Mo核酸的双重PCR方法,本研究根据ORFV的主要膜蛋白B2L基因(major envelope protein B2L gene)和Mo的膜蛋白P80基因(membrane protein P80 gene)序列,设计合成了2对特异性引物。结果显示,该方法可以同时扩增出ORFV的402 bp和Mo的700 bp特异性片段,而对其他常见病原的DNA模板均无扩增,显示出良好的特异性,该方法对ORFV和Mo的最低检出量分别为1.1×10~3copies/μL和3.47×10~3copies/μL,比单一PCR敏感10倍。对来自福建省部分羊场的883份病山羊(Capra hircus)临床样品进行检测,双重PCR检出ORFV阳性样品28份(阳性率为33.7%),Mo阳性样品33份(阳性率为39.8%),同时感染ORFV和Mo的阳性样品9份(阳性率为10.8%),检测结果与单一ORFV和Mo PCR结果一致。本研究所建立的双重PCR方法可用于ORFV和Mo的临床检测,为CE和MPGS的临床快速鉴别诊断和流行病学调查提供了有效的方法。
[Abstract]:Sheep infectious pustular virus (Orf virus,ORFV) is the pathogen of infectious pustular disease (Contagious ecthyma,CE), and mycoplasma pneumoniae (Mycoplasma ovipneumoniae,Mo) is the main pathogen of (Mycoplasma pneumonia of goats and sheep,MPGS. In recent years, the mixed infection of these two pathogens is increasing day by day. At present, a single PCR method is used to detect the two pathogens. In order to establish a double PCR method for the simultaneous detection of ORFV nucleic acid and Mo nucleic acid from the samples, the sequence of the main membrane protein (major envelope protein B2L gene of ORFV and the membrane protein P80 of Mo (membrane protein P80 gene was studied. Two pairs of specific primers were designed and synthesized. The results showed that the specific fragments of 402 bp of ORFV and 700 bp of Mo could be amplified simultaneously by this method, but the DNA templates of other common pathogens were not amplified, showing good specificity. The minimum detection levels for ORFV and Mo were 1.1 脳 10~3copies/ 渭 L and 3.47 脳 10~3copies/ 渭 L, respectively, which were 10 times more sensitive than single PCR. The (Capra hircus) clinical samples of 883 sick goats from some sheep farms in Fujian Province were detected. The positive rate of ORFV was 33. 7% (33. 7%) and 33 (39. 8%) by double PCR. 9 samples (10.8%) were positive for both ORFV and Mo, and the results were consistent with those of single ORFV and Mo PCR. The dual PCR method established in this study can be used for the clinical detection of ORFV and Mo, and provides an effective method for rapid differential diagnosis and epidemiological investigation of CE and MPGS.
【作者单位】：福建省农业科学院畜牧兽医研究所;
【基金】：福建省农业科学院科技创新项目(No.PC2017-9) 福建省科技创新平台建设项目(No.2014N2003-5) 国家重点研发计划(No.2016YFD0500906) 福建省农业科学院科技创新团队PI项目(No.2016PI-7)
【分类号】：S852.65

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