恒温实时荧光法快速检测不同样品中的单增李斯特菌  

Rapid Detection of Listeria monocytogenes in Different Samples by Real Time Fluorescence Isothermal Amplification

作　　者：张文敏[1] 石育娇 戚成 田明胜 朱智 殷荣永 钟少水 王大鹏[4] 何更生[5] 冯元琦 董庆利[1] ZHANG Wenmin;SHI Yujiao;QI Cheng;TIAN Mingsheng;ZHU Zhi;YIN Rongyon;ZHONG Shaoshui;WANG Dapeng;HE Gengshen;FENG Yuan qi;DONG Qingli(School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China;Shanghai Municipal Food and Drug Administration,Shanghai 200021,China;Shanghai Gemple Biotechnology Co.,Ltd.,Shanghai 201210,China;School of Agriculture and Biology,Shanghai Jiaotong University,Shanghai 200240,China;School of Public Health,Fudan University,Shanghai 200433,China;Guangzhou Deaou Biotechnology Co.,Ltd,Guangzhou 510663,China)

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093 [2]上海食品药品监督管理局,上海200021 [3]上海捷易生物科技有限公司,上海201210 [4]上海交通大学农业与生物学院,上海200240 [5]复旦大学公共卫生学院,上海200433 [6]广州迪澳生物科技有限公司,广州510663

出　　处：《食品与生物技术学报》2019年第5期44-50,共7页Journal of Food Science and Biotechnology

基　　金：国家重点研发计划(2018YFC1602902);国家“十二五”科技支撑计划项目(2015BAK36B04);国家自然科学基金项目(31271896)

摘　　要：为实现乳制品中、临床病人腹泻物中单增李斯特菌的快速检测,选用外引物F3和B3、内引物FIP和BIP作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)的特异性引物,采用便携式荧光恒温扩增仪作为检测平台,选取单增李斯特菌标准菌株进行基因组DNA灵敏度和最低检测限测定;选用人工污染的15份乳制品样品和20份腹泻样品进行适用性实验;以上样品同时利用国标法GB4789.30-2010进行菌落计数。结果表明:恒温实时荧光法对纯培养的单增李斯特菌基因组DNA、乳制品和腹泻样品中的单增李斯特菌基因组DNA灵敏度均达到10~2CFU/mL、检测限均达到10~3 CFU/mL;阴性样本出现假阳性的概率分别为0、0.03%和0;单增李斯特菌污染浓度在检出限以上的阳性样本检出率分别为100%、99%和92%。研究表明恒温实时荧光法的检测结果与传统国标培养结果基本一致,恒温实时荧光法适用于乳制品中和临床腹泻样本中单增李斯特菌的快速检测。In order to detect the Listeria monocytogenes in dairy products and clinical diarrhea rapidly,the outer primer F3 and B3,inter primer FIP and BIP were chosen as the loop-mediated isothermal amplification(LMAP)specific primers.Then a portable thermostat fluorescence detector was used as the detection platform,while the sensitivity and detection limit on genomic DNA were also determined with a standard strain of Lis.monocytegenes;Artificially contaminated 15 dairy products and 20 clinical diarrhea were served to measuring the applicability of this assay,and a Chinese national standard method(GB4789.30-2010)was used at the same time.The results showed that the sensitivity and detection limit respectively reached to 102 CFU/mL and 103 CFU/mL,while the probabilities of false positive were 0,0.03 and 0,the positive detection rate were 100%,99%and92%.The results were essentially in agreement with the culture method of GB4789.30-2010,indicating that this real time fluorescence isothermal amplification assay is suitable for the rapid detection of Lis.monocytegenes in dairy products and clinical diarrhea.

关 键 词：恒温实时荧光法 快速检测 单增李斯特菌 

分 类 号：R37[医药卫生—病原生物学]