益生菌制品中植物乳杆菌ST-Ⅲ快速定性、定量检测方法的建立  

Development on rapid qualitative and quantitative detection methods for Lactobacillus plantarum ST-Ⅲ in probiotics products

作　　者：陈臣[1] 任婧[1] 艾连中[1] 刘振民[1] 

机构地区：[1]光明乳业股份有限公司研究院/乳业生物技术国家重点实验室,上海200436

出　　处：《南方农业学报》2012年第10期1443-1446,共4页Journal of Southern Agriculture

基　　金：科技部“十二五”科技支撑计划项目(2012BAD28B08);科技部“973”计划项目(2012CB723706);科技部农业成果转化项目(2010GB2C000143)

摘　　要：【目的】建立快速定性、定量检测益生菌产品中植物乳杆菌菌株ST-Ⅲ的方法,为其实际应用提供参考。【方法】根据植物乳杆菌菌株ST-Ⅲ与NCBI库中3株已知序列的植物乳杆菌全基因组序列差异,设计菌株特异性引物,进行菌株特异性PCR验证和定性检测;然后建立荧光定量PCR,并检测益生菌制品中菌株ST-Ⅲ含量。【结果】设计的引物具有良好的菌株特异性;经PCR扩增,含有ST-Ⅲ的检验样本出现预期特征条带,与预期目的片段(242bp)相近。建立的荧光定量PCR标准曲线方程为y=-3.369lgx+39.84,R2为0.9990,符合Real-time PCR定量的要求,其检测限为2.85×103CFU/mL。经检测,样品中植物乳杆菌ST-Ⅲ的数量分别为3.25×105、1.28×106、4.55×106CFU/mL。【结论】建立的植物乳杆菌菌株ST-Ⅲ定性、定量检测方法快速灵敏、测定时间短,可以在实际生产中应用。[Objective ]In order to provide references for the reasonable use of L. plantarum ST-III, rapid qualitative and quantitative methods for ST-Ill detection in probiotics products were established through this research. [Method]The differences in the whole genome sequences between ST-III and the other three Lactobacillus plantarum strains accessed from the NCBI database were compared and the strain specificity primers of ST-III was designed accordingly in order to carry out strain-specific PCR analysis and fluorescent quantitative real-time PCR detection. The established real-time PCR method was used to detect the quantity of ST-Ill in probiotics products. [Result]The designed primers had good specificity for ST-III strain. The size of the expected specific band presented in the ST-Ill samples using PCR amplifica- tion was similar to that of the aiming band. The curve of established fluorescent quantitative real-time PCR was y= -3.3691gx＋39.84 （R2=0.9990）, which could demand the usage of real-time PCR, and its detection limit was 2.85×103 CFU/mL. By this detection method, the quantity of ST-III in probiotics products was 3.25×105, 1.28×106, and 4.55×106 CFU/mL, respectively. [ Conclusion ]The established methods for ST-III detection were rapid, accurate, and time- saving, therefore it could be used in relevant production processes.

关 键 词：益生菌 植物乳杆菌ST-Ⅲ PCR REAL-TIME PCR 定性定量检测 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]