乳和乳制品中肺炎克雷伯氏菌PCR-DHPLC检测新技术建立.PDF

微生物学通报 DEC 20, 2010, 37(12): 1805−1810 Microbiology China © 2010 by Institute of Microbiology, CAS tongbao@ 简 报 乳及乳制品中肺炎克雷伯氏菌 PCR-DHPLC 检测新技术的建立 1 2* 1 1 1 1 1 杨春华 曹际娟 钟毅 石磊 陈庆富 马欣欣 龚斐 (1. 江西出入境检验检疫局 江西 南昌 330002) (2. 辽宁出入境检验检疫局 辽宁 大连 116001) 摘 要: 为了应用PCR 结合变性高效液相色谱(DHPLC)技术建立乳品中肺炎克雷伯氏菌的快速检 测方法, 根据肺炎克雷伯氏菌 16S−23S rRNA 特异基因序列的特点设计特异性引物, PCR 扩增的产 物经DHPLC 技术进行快速检测。以肺炎克雷伯氏菌等 57 株参考菌株做特异性试验; 将肺炎克雷 伯氏菌菌株稀释成不同梯度, 做灵敏度试验。试验结果表明该方法具有很好的特异性, 灵敏度较 高, 检测低限可达到 100 CFU/mL, 可以快速、准确检测肺炎克雷伯氏菌, 是乳及乳制品中致病菌 快速检测的新技术。 关键词: DHPLC, 肺炎克雷伯氏菌, 16S−23S rRNA Construction of Identification of Klebsiella pneumoniae in Milk and Diary Product with PCR-DHPLC YANG Chun-Hua1 CAO Ji-Juan2* ZHONG Yi1 SHI Lei1 CHEN Qing-Fu1 MA Xin-Xin1 GONG Fei1 (1. Jiangxi Entry-Exit Inspection Quarantine Bureau of P.R.China, Nanchang, Jiangxi 330002, China) (2. Liaoning Entry-Exit Inspection Quarantine Bureau of P.R.China, Dalian, Liaoning 116001, China) Abstract: To identify the 16S−23S rRNA gene in Klebsiella pneumoniae , a PCR-DHPLC assay was performed in this study. Primers specific for the 16S−23S rRNA gene of Klebsiella pneumoniae in diary were selected to conduct the DHPLC assays. The specific testing was performed with Klebsiella pneu- moniae and 57 strains, and various grads of Klebsiella pneumo