地高辛标记探针检测鼻气管鸟杆菌的研究与应用

Study on Digoxigenin-labeled Nucleic Acid Probe for detection of Ornithobacterium rhinotracheale and its application

【目的】建立鼻气管鸟杆菌(Ornithobacteriumrhinotracheale,ORT)快速、准确的检测方法。【方法】利用PCR扩增鼻气管鸟杆菌16 S rRNA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。

[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 sensitivi- ty 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.