| 摘要: |
| 【目的】 建立鼻气管鸟杆菌（Ornithobacterium rhinotracheale,ORT）快速、准确的检测方法。【方法】 利用PCR扩增鼻气管鸟杆菌16 SrRNA基因的671 bp特异性片段，经回收纯化后用地高辛标记,建立了地高辛标记探针诊断ORT的方法。【结果】 特异性试验表明，地高辛标记探针可与ORT不同血清型参考菌株的核酸发生特异性杂交，而与鸡大肠杆菌、副鸡嗜血杆菌、鸡白痢沙门氏菌、禽多杀性巴氏杆菌的核酸杂交均为阴性；敏感性试验结果表明，地高辛标记探针对ORT的最低检出量为100 pg/μL；利用该探针对分离菌株和疑似病料进行检测，结果均与PCR检测结果一致。【结论】 建立了地高辛标记探针检测ORT的方法，为ORT的诊断提供了一种敏感性高、特异性强、简便且易行的方法。 |
| 关键词: 鼻气管鸟杆菌 地高辛标记探针 杂交检测 |
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| 基金项目:山东省自然科学基金项目（Y2006D10） |
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| Study on Digoxigenin labeled Nucleic Acid Probe for detection of Ornithobacterium rhinotracheale and its application |
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| Abstract: |
| 【Objective】 A method was optimized to detect ORT (Ornithobacterium rhinotracheale) more quickly and accurately.【Method】 The 671 bp specificity fragment was amplified by PCR from 16 S rRNA in ORT and through reclamation and cleansing this specificity fragment was labeled by Digoxigenin.A novel DIG Labeled DNA probe method was established for detecting the ORT.【Result】 From specificity test,it showed that this probe can develop specificity nucleate hybridization with different serotypes of ORT reference strains,but not in chicken E.coli,Haemophilus paragallinarum,Salmonella Pullorum and Avian pasteurella multocida.It indicated that this probe could detect the lowest of 100 pg/μL by sensitivity test.The isolation strains and a total of 211 doubtful samples were detected on the basis of this probe,and the result was identical in PCR.【Conclusion】 Digoxigenin labeled nucleic acid probe for detection of ORT was optimized and this research could provide a highly sensitive,convenient method for diagnosing ORT. |
| Key words: Ornithobacterium rhinotracheale Digoxigenin-labeled nucleic acid probe dot-blot hybridization |
