中国食品学报2011，Vol.11Issue(3)：138-143,6.
单核细胞增生李斯特菌荧光定量PCR检测试剂盒的研制
Development of a Real-time PCR Detection Kit for Listeria Monocytogenes
摘要
Abstract
Objective: The purpose of this study was to develop a Real-time PCR detection kit for the rapid detection of Listeria monocytogenes. Methods: The Taqman probe and primers for a Real-time PCR detection kit were designed according to the conserved gene hly of L. monocytogenes based on the previous study in our laboratory. The optimization of the kit components and the evaluation of its performance such as its specificity, sensibility, reproducibility and stability were carried out in this study. Results: A Real-time PCR detection kit was successfully developed in this study, which was used to specifically identify 8 reference strains of L. monocytogenes, while the strains from other species of Listeria and non-Listeria strains were not amplified with this kit. The detection limit of the kit was 3.94 fg/reaction with L. monocytogenes genomic DNA, i.e., 1.34 CFU/reaction. The average inter-assay CV％ of the kit was 2.2％ and the intra-assay CV％ was 1.9％, which showed a good reproducibility. The repeated frozen-thaw had no significant influence on the activity of this kit. Besides, this kit worked well after being stored at -20 ℃ for 1 year away from light. Conclusion: The Real-time PCR detection kit developed in this study is suitable for the rapid detection of L.monocytogenes for its high specificity, high sensitivity, good reproducibility and strong stability.关键词
单核细胞增生李斯特菌/荧光定量PCR/检测试剂盒Key words
Listeria monocytogenes/ Real-time PCR/ detection kit引用本文复制引用
张丹丹,龙飞,王大鹏,施春雷,史贤明..单核细胞增生李斯特菌荧光定量PCR检测试剂盒的研制[J].中国食品学报,2011,11(3):138-143,6.基金项目
"十一五"国家科技支撑计划项目(2009BADB9B01) （2009BADB9B01）
科技部国际合作项目(2011DFA31220,2009DFA31770) （2011DFA31220,2009DFA31770）
上海市科学仪器及化学试剂项目(10142201300,09142201200) （10142201300,09142201200）
