叠氮溴化丙锭结合实时荧光定量PCR技术检测发酵乳中植物乳杆菌P-8活菌数  

Detection of viable Lactobacillus plantarum P-8 in fermented milk using propidium monoazide combined with real-time fluorescence quantitative PCR

作　　者：韩之皓 郭帅 黄天 郑岩 王月娇 白梅[1] 王记成[1] 张和平[1] HAN Zhihao;GUO Shuai;HUANG Tian;ZHENG Yan;WANG Yuejiao;BAI Mei;WANG Jicheng;ZHANG Heping(Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Key Laboratory of Dairy Products Processing,Ministry of Agriculture,Inner Mongolia Agricultural University,Huhhot 010018,China)

机构地区：[1]内蒙古农业大学乳品生物技术与工程教育部重点实验室农业部奶制品加工重点实验室,内蒙古呼和浩特010018

出　　处：《食品与发酵工业》2019年第1期183-189,共7页Food and Fermentation Industries

基　　金：国家奶牛体系建设项目(CARS-36);内蒙古自治区科技计划重点项目(201702070)

摘　　要：采用叠氮溴化丙锭(propidium monoazide,PMA)结合实时荧光定量PCR对乳制品中活菌DNA进行定量分析,建立一种快速而准确检测发酵乳品中植物乳杆菌P-8(Lactobacillus plantarum P-8)活菌数的新方法。通过对影响PMA作用的浓度、暗孵育和曝光时间等因素进行试验,确定最佳PMA处理方案。结果表明:L. plantarum P-8经80℃处理60 s,即为膜损伤菌;当PMA质量浓度为40μg/m L,暗孵育时间10 min,曝光时间为20 min时,PMA既不影响活菌DNA的PCR扩增,又能渗透进入细胞膜受损的死菌并抑制其PCR扩增;通过制备L.plantarum P-8质粒标准品并建立标准曲线,其表现出良好的线性关系,相关系数(R2)为0. 992 9,最低检测限为103CFU/m L,特异性良好。该方法为完善发酵乳产品益生菌活菌数的检测奠定了基础。Propidium monoazide (PMA) was combined with real-time fluorescent quantitative PCR to detect DNA of living bacteria in milk products.A new,fast,and accurate method to detect Lactobacillus plantarum P-8 in fermented dairy products was developed.Influencing factors,such as concentrations,dark incubation,duration of illumination were determined to establish the optimal PMA treatment scheme.The results showed that after treating L.plantarum P-8 at 80℃ for 60s,they became membrane-damaging.When 40ug/mL PMA was used for 10min dark incubation,followed by espousing for 20min,PMA did not influence the living bacteria's DNA amplification.In addition,it inhibited DNA amplification of dead cells.A standard curve with a good linear relationship was set up based on L.plantarum P-8 plasmid standards.The relative coefficient of the curve was 0.9929,and the lowest detection limit was 10^3CFU/mL with good specificity.This method laid a foundation for improving the detection of viable probiotics in fermented milk products.

关 键 词：植物乳杆菌 叠氮溴化丙锭(propidium monoazide PMA) 实时荧光定量PCR 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]