实时荧光PCR法鉴定食品中双歧杆菌  

Identification of Bifidobacterium Strains in Foods by Real-Time PCR

作　　者：肖其胜[1,2] 杨捷琳[2] 杨惠琴[2] 丁卓平[1] 何宇平[2] 

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海出入境检验检疫局,上海200135

出　　处：《食品科学》2016年第20期177-182,共6页Food Science

基　　金：上海市科委长三角联合科技攻关项目(14495810200);国家质检总局项目(2015IK227)

摘　　要：根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法.对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25 份标示含双歧杆菌样品进行检测.结果表明：该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增.双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到104 CFU/mL.重复性测试表明相对标准偏差小于1%.同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好.对25 份市售实际样品进行测试,有5 份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分.本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌.A real-time PCR method was developed to identify Bifidobacterium in foods. Specific primers and probes targetingthe conserved region of the 16S rRNA gene of Bifidobacterium were designed. The specificity, sensitivity, reproducibility andsystem anti-interference ability of the proposed method were verified. Twenty-five commercial products labeled “containingBifidobacterium” were tested by the real-time fluorescence PCR assay. Results showed that the developed method washighly specific for Bifidobacterium detection. The detection limits were 2 pg using eight species of Bifidobacterium as thetemplates, and the relative detection limits were 104 CFU/mL. Standard deviations and relative standard deviations （RSDs）of Ct values for five DNA concentrations were all in the acceptable range. The Ct values were not affected by a mixture ofBifidobacterium adolescentis ATCC15703 and Escherichia coli ATCC25922 at both the culture level and genomic DNAlevel, which showed that the fluorescence PCR method has good anti-interference ability. Five samples were detectedwithout Bifidobacterium. Thus, considering its high specificity, sensitivity and repeatability, the real-time PCR could be usedto rapidly and accurately identify Bifidobacterium in foods.

关 键 词：实时荧光聚合酶链式反应 双歧杆菌 16S核糖体RNA 鉴定 

分 类 号：TS207.4[轻工技术与工程—食品科学]