北方某地区牛布菌病流行病学调查及布鲁菌多重pcr检测方法的建立.pdfVIP

摘 要 为了解北方某地区牛群中布鲁菌病的疫情，采取更为有效的防控措施，本文利用 虎红平板凝集试验（RBT ）、试管凝集试验（SAT ）和间接酶联免疫吸附试验（I-ELISA ） 随机对该地区牧场及散养牛群进行了布鲁菌病流行病学调查，同时对以上三种方法的 检测水平进行了比较；根据 GenBank 已发表的布鲁菌基因序列计合成三对特异性引 物，建立了同时检测布鲁菌属及牛、羊种布鲁菌的诊断鉴别性多重 PCR 方法，并对 其进行了初步应用。 用上述三种方法随机对北方某地区牧场和散养牛的 950 份血清进行布鲁菌病血 清抗体检测，表明该地区布鲁菌病流行情况较为严重，但不同群体阳性率差异较大， 牧场阳性率最高达 50%，最低阳性率仅为4.6% ，种牛场未检出阳性血清，散养奶牛 阳性率为39.3% ；RBT 检出阳性血清 105 份，阳性率为 15.8%，SAT 检出阳性血清 128 份，阳性率为 19.2%，I-ELISA 检出阳性血清262 份，阳性率为27.6% ，结果表 明I-ELISA 阳性率最高，SAT 次之，RBT 最低。 所建立多重PCR 方法的敏感性可达100pg 左右，且具有很好的特异性与重复性， 对7 株参考株葡萄球菌26001 等的PCR 扩增均为阴性，初步临床样品应用表明该方 法可用于乳样、阴道及粪便棉拭的检测。 关键词：布鲁菌病；流行病学调查；多重PCR ；应用 Bovine Brucellosis Serological Investigation in a Region of North China and Formation of Multi-PCR Detecting for Brucellosis Abstact To understand the situation of brucellosis in a region of north China and apply the more effective precautions, this study applied epidemiological survey stochastically in the grazing farms and distributive cattle groups in this region, using Rose Bengal Precipitation Test(RBT),Serum Agglutination Test(SAT) and Indirect Enzyme Linked Immunosorbent Assay(I-ELISA), and compared the three experiments, respectively. Based on the brucellosis gene sequence published on GenBank, specialized primer was synthesized, multi-PCR identification and diagnosis of Brucella and brucellosis on cows and sheep were established and primary application test was already conducted. Stochastically using the three methods above, 950 serum samples from the grazing farms and distributive cattle groups in a region of north China were inspected. Results showed that the epidemic situation of brucellosis is relatively serious. However, positive