多重PCR检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体

A Multiplex PCR Method for Detecting Brucella,Chlamydia psittaci and Coxiella burnetii of Dairy Cows

为了建立同步检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体感染的多重PCR方法,根据GenBank上具有属间特异性的布鲁菌.Bp26基因、鹦鹉热衣原体23S rRNA基因和贝纳氏柯克斯氏体IS1111a基因,利用Primer premier 5.0软件各设计1对特异性引物。通过优化PCR反应体系和扩增条件,建立了能够同时检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体的多重PCR方法。该方法具有较好的特异性和可重复性,对3种基因单重PCR检测敏感性均达到3.1×10~2拷贝·反应^-1,对3种病原基因同步检测的敏感性能达到3.1×10~3拷贝·反应^-1。利用该方法对采自不同流产牛的抗凝全血、血清、流产胎儿及奶液等172份临床样品进行检测,检测到布鲁菌感染阳性样品53份,鹦鹉热衣原体感染阳性样品2份,贝纳氏柯克斯氏体感染阳性样品10份,且检测到布鲁菌和鹦鹉衣原体混合感染阳性样品2份,布鲁菌和贝纳氏柯克斯氏体混合感染阳性样品2份,尚未检测到3种病原混合感染的阳性样品。结果表明,建立的多重PCR方法可以用来对奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体进行同步、快速、灵敏的检测。

Brucella,Chlamydia psittaci and Coxiella burnetii are three important zoonotic pathogens known to infect dairy cows.The aim of this study was to develop a multiplex PCR method for the simultaneous detection of them.Three genus-specific genes sequences were selected according to the GenBank,including Bp26 of Brucella spp.,23S rRNA of Chlamydia psittaci and IS1111a of Coxiella burnetii spp.And three specific primers pairs were designed respectively.After optimizing PCR reactions and amplification conditions,the multiplex method successfully discriminated Brucella,Chlamydia psittaci and Coxiella burnetii of dairy cows.The method had better specificity and repeatability,the detection sensitivity of simplex method for the three genes both reached 3.1×10~2 copies ? reaction^(-1),the detection sensitivity of multiplex method for these reached 3.1×10~3 copies ? reaction^(-1).Using this multiplex method to detect 172 samples including bloods,serums,placentas and milk of different abortion cattle,53 Brucella positive samples,2Chlamydia psittaci and 9 Coxiella burnetii positive samples were detected out,2 co-infection of Brucella and Chlamydia psittaci and 2 co-infection of Brucella and Coxiella burnetii positive samples were detected out else,no co-infection positive samples of the three pathogens was detected out.The above results indicated this method can be used for simultaneously,rapidly and sensitively detecting Brucella,Chlamydia psittaci and Coxiella burnetii of dairy cows.