荧光定量PCR快速检测鼠疫耶尔森氏菌的实验研究

Rapid detection of Yersinia pestis with real-time PCR assay

目的 利用LightCycler建立一种简便、特异的荧光定量PCR检测方法 ,用于鼠疫耶尔森氏菌的快速检测。 方法 采用SYBRGreenI随机掺入法 ,以Roche试剂盒为标准 ,比较两公司的DNA聚合酶 ,用鼠疫菌 310 0 4建立荧光PCR反应体系 ;针对鼠疫耶尔森氏菌特异的染色体标识序列和质粒上F1抗原基因设计引物 ,检测其灵敏度和特异性 ,以盲测试验进行验证 ;在此基础上鉴定 2 75株鼠疫耶尔森氏菌 ,并测试该法检测脏器污染模拟标本的灵敏度。结果 建立以一个公司试剂为主较低成本的PCR反应体系 ;EV76的检测灵敏度可达每反应体系 1.6个菌 ;检测我国 18个生态型共计 2 75株鼠疫耶尔森氏菌及 2 8株非鼠疫菌株的PCR扩增结果表明 ,鼠疫菌株均出现特异的扩增结果 ,2 8株对照菌株均为阴性 ;肝、脾、肺模拟标本检测灵敏度可达每反应体系 4 0 0 0个菌。结论 该方法对于检测鼠疫耶尔森氏菌具有简便、快捷、高敏感性和特异性的特点 。

To establish a simple and specific real-time PCR method for the rapid detection of Y. pestis strains. By using Roche kit as standard, the amplification efficiency of different reagents were compared by Roche Light Cycler instrument for establishing real-time PCR reaction system. The primers were designed based on signature sequence of chromosomal DNA fragment and caf 1 gene in plasmid pMT1 of Y.pestis. And sensitivity and specificity assay were done with serial diluted Y. pestis EV76 strain and other bacterias. Then 275 Y. pestis strains were identified and the detection limits of spike viscera samples were tested. Real-time PCR reaction system was established using MBI reagents. The sensitivity was 1.6 bacterias per reaction. The detection results of 28 strains of non-Y.pestis and 275 Y.pestis strains showed the high specificity of this assay. For the spike samples, 4?000 bacteria per PCR reaction system could be detected. The PCR assay was rapid and specific for rapid identification of Y. pestis, available for rapid diagnosis in case of emergency and the the surveillance of the plague natural foci.