布鲁氏菌和鹦鹉热衣原体双重PCR检测方法的建立与应用  

Establishment and Application of a Duplex PCR Method for Detecting Brucella and Chlamydia psittaci

作　　者：彭武丽[1,2] 罗展荣 史茜[2] 员丽娟[2] 于学辉[2] 季新成[2] 

机构地区：[1]石河子大学生命科学学院 [2]新疆出入境检验检疫局 [3]中国人民解放军69016部队

出　　处：《中国畜牧兽医》2014年第4期7-11,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家质量监督检验检疫总局科研项目(2014IK239;2011IK017);质检公益专项(20111024)

摘　　要：为建立同时检测布鲁氏菌和鹦鹉热衣原体的双重PCR方法,本研究据GenBank上已发表的具有属间特异性的布鲁氏菌bp26基因和鹦鹉热衣原体23SrRNA基因,利用Primer Premier 5.0软件各设计1对特异性引物,扩增的目的片段长度分别为219和356bp。通过优化反应条件,建立了能同时检测布鲁氏菌和鹦鹉热衣原体的双重PCR方法。该方法具有较好的特异性和可重复性,对2种基因单重PCR检测敏感性均达到3.1×102拷贝/反应,双重检测的灵敏度为3.1×103拷贝/反应。利用该双重PCR方法对流产牛抗凝全血、血清、流产胎儿及奶液共172份临床疑似布鲁氏菌感染的样品进行检测,检测到布鲁氏菌阳性样品53份,鹦鹉热衣原体阳性样品2份,以上这2种病原的阳性检出率分别为30.8%和1.2%,且检测到2种病原混合感染的阳性样品2份,阳性检出率为1.2%。临床应用结果表明,该方法可用来对布鲁氏菌和鹦鹉热衣原体进行同步、快速、灵敏的检测。To establish a duplex PCR method for simultaneously detecting Brucella and Chlamydia psittaci, this study ob- tained two genus-specific gene sequences according to the GenBank, including bp26 of Brucella spp. and 23S rRNA of Chlamydia psittaci. Two pairs of specific primers were respectively designed. After optimizing the reaction condition, the du- plex method was established for simultaneously detecting Brucella and Chlarnydia psittaci. The method had better specificity and repeatability, the detection sensitivity of simplex method for each gene both could reach 3. 1 × 10^2 copies per reaction, the detection sensitivity of duplex method for each gene could reach 3.1 × 10^3 copies per reaction. Using this duplex method to detect 172 samples including bloods, sera, placentas of abortion cattle and milk suspected infecting Brucella in clinic,53 Brucella positive samples and 2 Chlamydia psittaci positive samples were detected out, the positive rates were 30.8% and 1.2%, separately. 2 co-infection positive samples were detected out else and the positive rate was 1.2%. The above results indicated that this method could be used for simultaneously, rapidly and sensibly detecting the two pathogens of Brucella and Chlarnydia psittaci.

关 键 词：布鲁氏菌 鹦鹉热衣原体 双重PCR 共扩增 

分 类 号：Q78[生物学—分子生物学]