【作者】 祝岩波； 徐增年； 魏世锦； 郑龙； 尤红煜； 孟钰榕； 刘福英； 梁晓亮； 王俊霞；

【Author】 ZHU Yan-bo;XU Zeng-nian;WEI Shi-jin;ZHENG Long;YOU Hong-yu;MENG Yu-rong;LIU Fu-ying;LIANG Xiao-liang;WANG Jun-xia;Department of key laboratory Animal Hebei Province, laboratory Animal Hebei Medical University;Baixiang Central Hospital;

【机构】 河北省实验动物重点实验室河北医科大学实验动物学部； 柏乡县中心医院；

【摘要】 目的建立快速检测多杀巴氏杆菌、支气管鲍特杆菌、支原体和肺炎克雷伯杆菌4种实验动物病原体的多重PCR方法。方法根据Genebank基因设计出特异性引物;经过多重PCR的优化,特异性和敏感性的检测,建立多重PCR体系。应用该PCR体系检测人工感染样本和实验动物的气管分泌物,并与传统方法做对比。结果多重PCR扩增出多杀巴氏杆菌(356 bp)、支气管鲍特杆菌(237 bp)、支原体(266 bp)和肺炎克雷伯杆菌(142bp)的目的条带。肺炎克雷伯杆菌敏感性为10 pg,多杀巴氏杆菌、支气管鲍特杆菌、支原体敏感性为1 pg,特异性检测未从其他病原菌中检测出目的条带。应用建立的多重PCR体系检测人工感染样本的不同组合,45只实验动物气管检测出15只多杀巴氏杆菌阳性,9只肺支原体阳性,但传统培养方法和血清学方法未检测出阳性标本。结论本文建立的多重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对多杀巴氏杆菌、支气管鲍特杆菌、支原体和肺炎克雷伯杆菌4种实验动物病原体的快速检测。更多还原

【Abstract】 Objective The aim of this study is to establish a multiplex polymerase chain reaction(PCR) assay forsimultaneous identification of four kands of laboratory animal pathogen which isPasteurella multocida、Bordetella bronchiseptica、Mycoplasma pneumonia and Klebsiella pneumoniae. Method Based on the Genebank genes designed specific primers; Throughthe optimization of multiple PCR, detection of specificity and sensitivity, establishing multiple PCR system. PCR systemapplication of the test samples of artificial infection and experimental animals tracheal secretions,is in accord with comparing withthe traditionalmethod.Result Multiple PCR amplifiesPasteurella multocida(356 bp),Bordetella bronchiseptica(237 bp),Mycoplasma pneumonia(266 bp) andKlebsiella pneumoniae(142 bp),sensitivity ofKlebsiella pneumoniaefor 10 pg, sensitivityofPasteurella multocida,Bordetella bronchiseptica and Mycoplasma pneumoniafor 1 pg, specificity was not detected from other pathogensobjective stripe application to establish system of multiplex PCR detection of artificial samples of different combinationof infection, 45 experimental animals trachea detect 15 Pasteurella multocidapositive and 9 Mycoplasma pneumoniapositive,whiletraditionalmethod and serololgicalmethod detect the negative for the specimens.Conclusions A simple,rapid, specific andhighly sensitive multiplex PCR system has been established and it is valuable for detectioncan forPasteurella multocida、Bordetella bronchiseptica、Mycoplasma pneumonia and Klebsiella pneumoniae.更多还原