流感嗜血杆菌、溶血嗜血杆菌和多杀巴斯德杆菌病多重荧光定量PCR检测方法的建立与初步应用  

Development and Application of a Multiple Real-time Quantitative PCR for Haemophilus influenzae,Haemophilus haemolyticus and Pasteurella multocida

作　　者：邢进[1] 冯育芳[1] 岳秉飞[1] 贺争鸣[1] 孙晓梅[2] 代解杰[2] 

机构地区：[1]中国食品药品检定研究院实验动物资源研究所,北京100050 [2]中国医学科学院/北京协和医学院医学生物学研究所树鼩种质资源中心,昆明650118

出　　处：《实验动物科学》2017年第3期38-42,共5页Laboratory Animal Science

基　　金：国家科技支撑计划项目(No.2014BAI01B01)

摘　　要：目的建立同时检测实验动物中的流感嗜血杆菌、溶血嗜血杆菌和多杀巴斯德杆菌的多重实时荧光定量PCR(qPCR)检测方法。方法分别根据三种病原菌的Hpd基因和KMT1基因设计特异性引物和Taqman探针,经特异性、敏感性和重复性验证,建立多重qPCR检测方法,并对822份实验动物呼吸道样本进行检测应用。结果所建立的多重qPCR方法与其他29种病原菌间无交叉反应,对三种病原菌的检测限可达到10个拷贝/μL。树鼩样品中溶血嗜血杆菌和多杀巴斯德杆菌的阳性率分别为10%(6/60)和21.7%(13/60),其他实验动物中均未检出上述三种病原菌。结论本研究所建立的多重qPCR检测方法,可快速检测实验动物中的上述三种呼吸道病原菌。Objective To develop a multiplex real-time quantitative PCR （qPCR） assay to detect Haemophilus influenzae, Haemophilus haemolyticus and Pasteurella multoeida in laboratory animals. Method We establish the multiple qPCR reaction system by specificity, sensitivity and reproducibility verification, according to Hpd gene of H. influenzae and H. haemolytieus, KMT1 gene of P. multoeida designed specific primers and Taqman probes respectively. Then, the 822 laboratory animal respiratory samples were detecting by the assay application. Result There was no cross reaction between the three pathogens and 29 others. The sensitivity of the multiple qPCR method detecting above three pathogens could reach 10 eopies/txL. Among the tree shrew specimens,the positive rate of H. haemolyticus and P. multocida were 10% （6/60） and 21.7% （13/60） respectively, but other animals were negative in three pathogens. Conclusion The multiple qPCR assay in this study that can quickly detect the above three kinds of respiratory pathogen in laboratory animals.

关 键 词：流感嗜血杆菌 溶血嗜血杆菌 多杀巴斯德杆菌 多重荧光定量PCR 实验动物 

分 类 号：Q95-33[生物学—动物学]