乳品中大肠杆菌PMA-qPCR活菌检测方法的建立  

Establishment of PMA-qPCR Assay for Detection of Viable E.coli in Milk

作　　者：盖冬雪 任洪林[1] 卢士英[1] 胡盼[1] 孟宪梅[2] 刘熙 宋德刚 金雯[1] 张嵩[1] 常江[1] 刘艳艳 柳增善[1] 

机构地区：[1]吉林大学人兽共患病研究所,人兽共患病教育部重点实验室,长春130062 [2]吉林工商学院,粮油食品深加工吉林省高校重点实验室,长春130000 [3]广泽乳业有限公司,长春130000 [4]山东省东营市利津县第二中学,东营257400

出　　处：《中国畜牧兽医》2016年第2期493-498,共6页China Animal Husbandry & Veterinary Medicine

基　　金：吉林省重点科技攻关项目(20140204065NY);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);粮油食品深加工省高校重点实验室项目(2014001)

摘　　要：本试验旨在建立一种乳品中大肠杆菌PMA-qPCR活菌检测方法。优化qPCR检测方法,探究菌浓度为1×108 CFU/mL的大肠杆菌活菌悬液、热致死菌悬液细胞数来确定不同的PMA剂量、暗孵育时间、曝光时间对死菌抑制效果的影响,确定最佳PMA处理方案。结果表明,qPCR检测可特异性扩增大肠杆菌,1×108 CFU/mL的大肠杆菌经90℃水浴30s全部致死后,采用10μg/mL的PMA暗孵育15min后冰上曝光10min为最佳处理方案,这种处理方案可最大程度抑制死细胞信号,而对活细胞几乎没有影响,样品中微生物初始浓度不低于1×108 CFU/mL时较稳定,得到标准曲线回归方程y=-3.356x+47.413,R2=0.9989,最低检测限为103 CFU/mL,加标样本检测结果与实际相符。该方法为利用PMA-qPCR检测食品中的活大肠杆菌杆菌奠定了基础。In order to detect viable E.coli in milk,a new PMA-qPCR method was established.The influences of different PMA concentration,dark incubation time,exposure time on dead bacteria inhibition effect were determined by detection of the cell numbers of viable and heat-killed E.coli suspensions at concentration of 1×108 CFU/mL through fluorescence quantitative PCR（qPCR）method.The results showed that qPCR assay could specifically detect E.coli,and the viable E.coli must be exposed to 90℃for 30 sin water bath to be lethal.The best treatment was 10μg/mL PMA with 15 min of dark incubation time and 10 min of exposure time.This treatment could inhibit dead cell signals to a largest extend,while had little impact on aviable cells.The stability of PMAqPCR assay was kept while the concentration of bacteria was more than 1×108 CFU/mL.The regression equation of standard curve was y=-3.356x＋47.413,R2=0.9989,the lowest detection limit was 103 CFU/mL.The result of adding assay was agreed with the actual situation.This study laid a foundation for using of PMA-qPCR to detect the viable E.coli in food.

关 键 词：大肠杆菌 叠氮溴化丙啶 qPCR 计数 

分 类 号：Q78[生物学—分子生物学]