摘要
本研究旨在建立溶血性曼氏杆菌与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两种病原的感染提供一种更加方便、快捷、准确的工具。根据溶血性曼氏杆菌lkt基因和多杀性巴氏杆菌sp6基因分别设计引物,优化条件后进行特异性和敏感性的评价,并对180份临床样本进行了检测。结果显示,溶血性曼氏杆菌和多杀性巴氏杆菌引物的最佳终浓度分别为10和20pmol/L。该方法的特异性较好,对其他无关病原不检出。对溶血性曼氏杆菌和多杀性巴氏杆菌的检测限分别为56和22pg,与独立PCR相同。该方法对临床样本的检测结果表明,溶血性曼氏杆菌和多杀性巴氏杆菌的检出率分别为51.67%和47.78%。研究结果表明,本研究建立的双重PCR诊断技术具有特异性好、敏感度高等特点,为临床上溶血性曼氏杆菌和多杀性巴氏杆菌的快速检测、鉴定以及混合感染的流行病学调查提供了有用的工具。
The purpose of this study was to establish a duplex PCR method for simultaneous detection of Mannheimia haemolytica and Pasteurella multocidain order to provide a rapid,accurate and convenient detection tool for the two pathogens.A set of primers was designed based on M.haemolytica lkt gene and P.multocida sp6 gene.Following optimization of PCR components and reaction profile,specificity and sensitivity of the assay were evaluated.Subsequently,a total of 180 clinical samples were tested by the duplex PCR.The results demonstrated that optimal final concentrations of primers were 10pmol/L for M.haemolyticaand 20pmol/L for P.multocida.The assay was highly specific for M.haemolyticaand P.multocida without cross reactions with other pathogens.The detection limits of the assay were determined to be 56pg and 22pg for M.haemolytica and P.multocida,respectively,which was as sensitive as single PCR methods.Detection of clinical samples using the duplex PCR demonstrated a 51.67% detection rate for M.haemolyticaand 47.78% detection rate for P.multocida.These results suggested that the developed duplex PCR assay was specific and sensitive,and will be useful for clinical detection,identification of M.haemolyticaand P.multocidaand epidemiological investigation of co-infection of the two pathogens.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2014年第6期624-629,共6页
Chinese Veterinary Science
基金
国家星火计划重大项目(2012GA810001-6)
