| 猪肺炎支原体显色原位杂交研究方法的建立 |
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| 引用本文： | 王占伟,熊祺琰,刘茂军,冯志新,白方方,邵国青.猪肺炎支原体显色原位杂交研究方法的建立[J].中国预防兽医学报,2012,34(3):214-218. |
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| 作者姓名： | 王占伟 熊祺琰 刘茂军 冯志新 白方方 邵国青 |
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| 作者单位： | 1. 江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014;南京天邦生物科技有限公司,江苏南京210012
2. 江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京,210014 |
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| 基金项目： | 国家自然科学基金(31001080);国家农业科技成果转化基金(2009GB2C100129);江苏省农业科技自主创新资金项目[cx(09)619];中国博士后科学基金面上资助项目(20090461075) |
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| 摘 要： | 为建立猪肺炎支原体(Mhp)显色原位杂交(CISH)定位检测方法,本研究利用地高辛标记的Mhp P36核酸探针和DAB/H2O2显色系统,对人工Mhp感染组、自然感染组和健康对照组的实验猪肺组织样品和临床样品进行显色原位杂交检测.结果显示,设计的地高辛标记探针针对Mhp具有特异性,而与其他的猪病原菌无交叉反应；检测结果可以通过显微镜观测到Mhp定位在支气管/细支气管黏膜表面.人工感染组、自然感染组和健康对照组的CISH结果与PCR检测结果一致,临床样品的CISH检测结果低于PCR检测结果.本研究建立的Mhp显色原位杂交法是一种集直观和敏感为一体的检测方法,可以用于Mhp的定位检测和致病机理研究.
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| 关 键 词： | 猪肺炎支原体 显色原位杂交 斑点杂交 检测 定位 |
Development of chromogenic in situ hybridization for Mycoplasma hyopneumoniae |
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| WANG Zhan-wei
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XIONG Qi-yan
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LIU Mao-jun
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FENG Zhi-xin
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BAI Fang-fang
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SHAO Guo-qing.Development of chromogenic in situ hybridization for Mycoplasma hyopneumoniae[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):214-218. |
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| Authors: | WANG Zhan-wei XIONG Qi-yan LIU Mao-jun FENG Zhi-xin BAI Fang-fang SHAO Guo-qing |
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| Institution: | 1(1.Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,National Center for Engineering Research of Veterinary Bio-products,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China; 2.Nanjing Teeh-bank Bio-industry Co.,Ltd,Nanjing 210012,China) |
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| Abstract: | To develop a chromogenic in situ hybridization(CISH) assay for the detection of Mycoplasma hyopneumoniae(Mhp) in pigs,CISH was performed by using a digoxigenin-labeled DNA probe specific for P36 gene of Mhp,and using 3,3-diaminobenzidine(DAB)/H2O2 for visualization.Lung tissue samples collected from experimentally infected pigs,naturally infected pigs and suspected infected pigs were detected with samples from healthy pigs as negative control.The results showed that the designed digoxigenin-labeled probe was specific for Mhp,which had no cross-reaction with other pig pathogens.Mhp located in bronchial and bronchial mucosal surface could be observed under microscope.The CISH test results of artificial infection group,natural infection group and control group were consistent with the results of PCR method,while the positive ratio of clinical samples detect by CISH method was lower than the result of PCR method.In conclusion,this research provides an intuitive and sensitive CISH method to detect Mhp,which would help to study the location of Mhp in vivo and the pathogenic mechanism of Mhp infection. |
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| Keywords: | Mycoplasma hyopneumoniae chromogenic in situ hybridization dot blot hybridization detection location |
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