丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

中国畜牧兽医

丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

王成龙，吴禹熹，冯旭飞，杨发龙

（西南民族大学生命科学与技术学院，四川成都 610041）

修回日期:2014-02-07 出版日期:2014-04-20 发布日期:2014-05-27
通讯作者: 杨发龙（1974—），男，青海人，博士，副教授，研究方向：病原微生物学。E-mail：yfalong@hotmail.com
作者简介:王成龙（1988—），男，吉林人，硕士生，研究方向：病原微生物学。
基金资助:
国家星火计划重大项目（2012GA810001-6）；西南民族大学研究生创新型科研项目（CX2013SZ74）。

Development and Application of a Duplex PCR Assay for the Detection of Mycoplasma mycoides Cluster and Pasteurella multocida

WANG Cheng-long, WU Yu-xi, FENG Xu-fei, YANG Fa-long

(College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China)

Revised:2014-02-07 Online:2014-04-20 Published:2014-05-27

摘要/Abstract

摘要： 本研究旨在建立丝状支原体簇和多杀性巴氏杆菌的双重PCR检测方法，从而为临床上同时检测这2类病原的感染提供一种更方便、快捷、准确的工具。本研究采用2对特异性检测丝状支原体簇和多杀性巴氏杆菌的引物，对PCR反应体系和反应条件进行了优化，并对双重PCR的特异性及敏感性进行了评价，随后采用该方法对52份临床样本进行了检测。结果显示，所建立的双重PCR方法能同时扩增丝状支原体簇成员和多杀性巴氏杆菌的DNA，而对来源于其他常见病原的DNA均无扩增；对丝状支原体簇和多杀性巴氏杆菌的最低检测限分别为24.8和28.9 pg；能成功地从临床样本中检测丝状支原体簇成员和多杀性巴氏杆菌。结果表明,本研究所建立的双重PCR方法具有很好的特异性和敏感性，为临床丝状支原体簇和多杀性巴氏杆菌感染的快速诊断、病原鉴定及流行病学调查提供了有效的方法。

关键词: 丝状支原体簇; 多杀性巴氏杆菌; 双重PCR

Abstract: The aim of this study was to establish a duplex PCR method for simultaneous detection of Mycoplasma mycoides cluster and Pasteurella multocida, so as to provide a rapid, accurate and convenient detection tool for these pathogens. Two pairs of primers which were specific to Mycoplasma mycoides cluster and Pasteurella multocida，respectively, were used to develop the method. Following optimization of PCR components and reaction profile, specificity and sensitivity of the assay were evaluated. A total of 52 clinical samples were tested using the duplex PCR. The results showed that the assay could specifically amplify Mycoplasma mycoides cluster and Pasteurella multocida, while no-target pathogens were not detected. The detection limits of the assay were determined to be 24.8 and 28.9 pg for Mycoplasma mycoides cluster and Pasteurella multocida, respectively. The duplex PCR could detect Mycoplasma mycoides cluster and Pasteurella multocida from clinical samples. These results suggested that the duplex PCR assay was specific and sensitive, and would be useful for clinical detection, identification and epidemiological investigation of Mycoplasma mycoides cluster and Pasteurella multocida.

Key words: Mycoplasma mycoides cluster; Pasteurella multocida; duplex PCR

王成龙，吴禹熹，冯旭飞，杨发龙. 丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用[J]. 中国畜牧兽医.

WANG Cheng-long, WU Yu-xi, FENG Xu-fei, YANG Fa-long. Development and Application of a Duplex PCR Assay for the Detection of Mycoplasma mycoides Cluster and Pasteurella multocida[J]. China Animal Husbandry & Veterinary Medicine.

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