泡菜中乳酸菌活菌的EMA结合定量PCR检测方法的建立  

Establishment of Ethidium Monoazide and Quantitative PCR Method to Detect Viable Lactic Acid Bacteria(Lactobacillus plantarum) in Kimchi

作　　者：石慧[1,2] 程国灵[1] 翟百强 黄昆仑[1,2] 罗云波[1,2] 戴蕴青 田文莹[1] 许文涛[1,2] 

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083 [2]农业部农产品贮藏保鲜质量安全风险评估实验室(北京),北京100083 [3]北京福德安科技有限责任公司,北京100085

出　　处：《农业生物技术学报》2013年第3期373-378,共6页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划项目(No.2011BAK10B02);863科技攻关项目(No.2012AA101606)

摘　　要：泡菜中乳酸菌(Lactobacillus plantarum)活菌的数量是评价其营养价值的重要指标。DNA染料(Ethidium bromide monoazide,EMA)结合定量PCR(EMA-qPCR)是一种定量活细胞快速有效的方法。本研究建立了快速精准的检测泡菜中乳酸菌活菌的EMA-qPCR方法。对于乳酸菌死菌,EMA浓度为10μg/mL时,ΔCt值(经EMA处理-未经EMA处理)最大,而乳酸菌的ΔCt值在不同EMA浓度下(10,25,50和100μg/mL)没有显著差异(P>0.05)。并且,随着10μg/mL EMA的光活化时间由0min增加到20min,ΔCt值也随之增加,但是这种现象没有在活细胞中产生,所以EMA处理不会影响活细胞计数。因此最适宜的EMA处理条件(10μg/mL,光照20min)可以有效的区分乳酸菌活细胞和死细胞。在此条件下EMA-qPCR方法(活乳酸菌数量分别为109、108、107、106、105和104CFU/mL)与平板计数法的检测结果相关性良好(R2=0.999),PCR效率为104%。由于泡菜中乳酸菌随着发酵时间延长会出现亚致死性损伤,EMA-qPCR方法会低估活菌的数量,在检测前将菌体在MRS培养基中孵育30min会完全消除这种偏差。本实验建立的EMA-qPCR方法,可成功地检测不同发酵时间泡菜中乳酸菌活菌的数量。通过摸索适宜条件,这种检测死活菌的方法可应用到更多的食品与环境检测中。Kimchi is a kind of traditional fermented vegetable, and it is also one well-know beneficial food. The number of viable lactic acid bacteria （LAB） is an important indicator to assess the nutritional value of kimchi. Ethidium monoazide in combination of quantitative PCR （EMA-qPCR） has been considered as a rapid and effective method to enumerate viable cell. In this study, EMA-qPCR method was established to detect viable LAB（Lactobacillus plantarum） rapidly and precisely in kimchi. For non-viable LAB, the maximum ACt （with EMA - without EMA） was achieved at an EMA concentration of 10 μg/mL, and there were no significant differences （P〉0.05） in the ACt values for LAB treated with different EMA concentrations of 10, 25, 50 and 100 μg/mL. Moreover, the ACt increased significantly （P〈0.05） as the photoactivation time of EMA increased from 0 to 20 min at 10 Ixg/mL EMA. But this effect was not observed among viable LAB, so EMA treatment could not affect the enumeration of viable cells. Therefore, optimum EMA treatment （10 μg/mL and 20 min light activation） lead to effective discrimination between viable and dead LAB. Under these conditions, results from EMA-qPCR （viable LAB of 109, 108, 107, 106, 105 and 104 CFU/mL） correlated well with that of plate counting （R2=0.999）, and PCR efficiency reached to 104%. Due to sublethally injury of LAB with fermentation proceeding, EMA could penetrate into sublethal injured cell, and EMA-qPCR method consequently underestimated cell counts. Incubating cells in MRS medium for 30 min before detection could offset this error. That because incubation for suitable time could recover the sublethally injured cells but could not increase cell number. That indicated that EMA-qPCR could not only discriminate viable and non-viable cells, but also detect the sublethally injured cells. The viable LAB counts detected by EMA-qPCR increased with fermentation time increasing in the early stage of kimchi fermentation. As fermentation proceeded, lactic acid prod

关 键 词：泡菜 乳酸菌 活细胞 ETHIDIUM BROMIDE monoazide 聚合酶链式反应 

分 类 号：S858.23[农业科学—临床兽医学]