EMA-PCR法快速检测啤酒中腐败短乳杆菌  

Development of an Ethidium Bromide Monoazide-Polymerase Chain Reaction Assay for Raid Detection of the Beer Spoilage Bacterium Lactobacillus brevis

作　　者：马艳琳[1,2] 徐振波[3] 刘君彦[3] 汪东风[1] 邓阳[1,2] MA Yanlin;XU Zhenbo;LIU Junyan;WANG Dongfeng;DENG Yang(College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266100, China;School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003 [2]啤酒生物发酵工程国家重点实验室,山东青岛266100 [3]华南理工大学食品科学与工程学院,广东广州510641

出　　处：《食品科学》2017年第8期271-276,共6页Food Science

基　　金：中国博士后科学基金资助项目(2015M582063;2014T70810);南昌大学食品科学与技术国家重点实验室开放基金资助项目(SKLF-KF-201415)

摘　　要：将叠氮溴乙锭(ethidium bromide monoazide,EMA)与聚合酶链式反应(polymerase chain reaction,PCR)技术相结合,以酒花耐受基因hor C为靶基因,用啤酒腐败短乳杆菌基因组DNA作为模板进行扩增。结果发现,在前处理过程中加入EMA,当终质量浓度小于20μg/m L时,对活的短乳杆菌中靶基因的扩增没有明显抑制作用;而当EMA终质量浓度为1.0μg/m L时可有效抑制10~5 CFU/m L短乳杆菌死细胞的扩增。本实验建立的EMA-PCR检测方法的灵敏度为10~4活细胞/m L酒液样品。验证实验结果表明,在13株乳酸菌中,建立的hor C特异性EMA-PCR能有效检测到其中的全部5株啤酒污染菌,同时可区分这5株菌的活/死细胞混合体系,降低检测过程中的假阳性。In this paper,a rapid method using conventional PCR after ethidium bromide monoazide(EMA)pretreatment is described for the detection of Lactobacillus brevis as a beer spoilage bacterium.The PCR amplification was carried out using the hop resistance gene horC as target gene and the genomic DNA from L.brevis as template.The results suggested that addition of EMA to a final concentration lower than20μg/mL for pretreatment did not strongly inhibit the PCR amplification of DNA derived from viable L.brevis cells,but it,when added to a final concentration of1.0μg/mL,completely inhibited the PCR amplification of DNA derived from105CFU/mL dead L.brevis cells.The detection limit(LOD)of EMA-PCR assay was found to be104CFU/mL beer sample for the horC gene.Moreover,the horC-specific EMA-PCR assay was applied to detect13strains of lactic acid bacteria,representing100%specificity with no false positive amplification observed for five beer spoilage lactic acid bacteria and enabling discrimination between the live and dead cells.Overall,the use of horCspecific EMA-PCR allows for a substantial reduction in the rate of false-positive results for potential beer spoilage L.brevis.

关 键 词：叠氮溴乙锭 聚合酶链式反应 啤酒腐败菌 短乳杆菌 酒花耐受基因 

分 类 号：TS261.1[轻工技术与工程—发酵工程]