灭菌乳中活阪崎肠杆菌PMA-PCR检测方法的建立  

Development of PMA-PCR Method for Detection of Viable Enterobacter sakazakii in Pasteurized Milk

作　　者：刘艳艳[1,2] 柳增善 卢士英[1,2] 任洪林 盖冬雪[1,2] 崔成 丁艳霞[1,2] 孟宪梅 于师宇 

机构地区：[1]人兽共患病研究教育部重点实验室,吉林大学人兽共患病研究所 [2]吉林大学动物医学学院,吉林长春130062 [3]吉林工商学院粮油食品深加工吉林省高校重点实验室,吉林长春130062 [4]福建出入境检验检疫局,福建福州350001

出　　处：《中国畜牧兽医》2014年第2期65-69,共5页China Animal Husbandry & Veterinary Medicine

基　　金：吉林省世行贷款农产品质量安全项目(2011-Y36);吉林省科技发展计划项目(201205054)

摘　　要：本试验旨在建立灭菌乳中活阪崎肠杆菌(Enterobacter sakazakii)的PMA-PCR检测方法。将DNA染料叠氮溴化丙锭(propidium monoazide,PMA)与聚合酶链式反应(polymerase chain reaction,PCR)技术结合,优化PMA最佳工作浓度和曝光时间,确定PMA-PCR区别死、活阪崎肠杆菌细胞的最佳条件。结果表明,活阪崎肠杆菌经100℃沸水处理20min可完全致死,PMA完全与死阪崎肠杆菌DNA共价交联并光解溶液中游离PMA的最佳曝光时间为15min,完全抑制死阪崎肠杆菌PCR扩增的最小PMA浓度为5μg/mL,不抑制活阪崎肠杆菌PCR扩增的最大PMA浓度为15μg/mL。经PMA处理后,在含有不同比例的死、活阪崎肠杆菌混合液中检测到活阪崎肠杆菌的最低检测限为40CFU/mL,在灭菌乳样品中检测到活阪崎肠杆菌的最低检测限为100CFU/mL。该方法为利用PMA-PCR检测食品中的活阪崎肠杆菌奠定了基础。In order to detect viable Enterobacter sakazakii in pasteurized milk, a new method was established by combination of propidium rnonoazide （PMA） and polymerase chain reaction （PCR）. PMA concentration and exposure time were optimized to find optimal conditions for distinguishing between dead and viable E. sakazakii. The results showed that to kill the viable E. sakazakii must be exposed to 100 ℃ for 20 min in water bath. The optimum light exposure time to intercalate the DNA of dead E. sakazakii and to photolyze the free PMA in solution was I5 min. The minimum concentration of PMA to completely inhibit the PCR amplication of dead bacterium was 5 μg/mL. The maximum concentration of PMA did not inhibit the PCR amplifica- tion of viable bacterium was 15μg/mL. After PMA treatment,viable E. sakazakii in a mixture containing different proportions of dead and viable bacterium could be selectively detected by PCR, and the determination limit was 40 CFU/mL. In the pasteur- ized milk,the determination limit was 100 CFU/mL. This study laid a foundation for use of PMA-PCR to detect the E. sakaza- kii in the food.

关 键 词：PMA PCR 阪崎肠杆菌 灭菌乳 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]