Application of 16S rDNA fingerprint on the strain identification and traceability isolated from yogurts
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摘要: 近年来,微生态制剂日益受到市场关注。市售微生态食品以益生菌酸奶或乳酸饮料为主,其中活的乳酸菌含量及菌株特性是决定微生态制剂质量的关键。目前常用的菌种鉴定技术包括生理生化方法或PCR方法,前者工作量大周期长,后者需要专业人员操作,且难以区分死活细菌。本文选取了11种市售的乳酸菌产品,采用MC、MRL、TPY等乳酸菌专用培养基进行乳酸菌计数及菌种分离,进一步用荧光定量PCR方法验证了产品中常规培养法难以分离的双歧杆菌,在此基础上,采用Riboprinter基因指纹图谱分析仪,对分离自11种酸奶样品的16株乳酸菌菌进行了核酸水平的鉴定,建立了16S rDNA聚类分析分子图谱,方便并简化对微生态产品的质量进行"物证相符"的考证,初步分析了目前市场上市售酸奶所用菌种的种属相关性。
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关键词:
- 乳酸菌; /
- Riboprinter全自动微生物基因指纹鉴定系统; /
- 荧光定量PCR;
Abstract: Microecological products are paid more and more attention.Most commercial microecological products on local market are the yogurts which contain lactic acid bacteria.The common technology of strain identification are the physiological and biochemical test or the application of PCR with some defects such as time-consuming or need specialized person.We used Riboprinter to complete the subspecies level of identification of 16 lactobacillus bacterias isolated from 11 yogurt samples based on the separation and purification of the bacterias.Not only chose the optimization of the culture conditions, but also established the “Traceability File” used for the quality research.What’s more, the use of the 16s rDNA fingerprint analysis and real time PCR helped to confirm the verification of the results from the colony identification. -
[1] 董相军, 张彩霞.我国微生态制剂的概述[J].科技咨询, 2007 (18) :241-242. [2] Ger T Rijkers, Stig Bengmark, Paul Enck, et al.Guidance for substantiating the evidence for beneficial effects of probiotics:current status and recommendations for future research[J].The Journal of Nutrition, 2010, 140 (3) :671S-676S.
[3] 刘斌, 黄少磊, 刘彦民, 等.分子标记技术应用于乳酸菌分类鉴定的研究进展[J].中国微生态学杂志, 2012, 24 (7) :35-38. [4] Matthieu Legendre, Ste’phane Audic, Olivier Poirot, et al.mRNA deep sequencing reveals75new genes and a complex transcriptional landscape in Mimivirus[J].Genome research, 2010, 20:664-674.
[5] Valerie Coeuret, Micheline Gueguen, Jean Paul Vernoux.Numbers and strains of lactobacilli in some probiotic products[J].International Journal of Food Microbiology, 2004, 97 (2) :147-156.
[6] 林莹, 代道芳, 辛志平, 等.传统培养结合分子生物学法分析传统发酵食品中乳酸菌的多样性中国酿造[J].2011, 2:52-54. [7] 柴丽, 董良仓.乳酸菌基因组学研究进展[J].检验医学与临床, 2012, 9 (2) :214-215. [8] 凌代文主编.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社, 1999:84-138. [9] Riboprinter microbial detection system user Manual[M].Dupont Qulicon, 2002:23-46.
[10] Matsuki T, Watanabe K, Tanaka R, et al.Rapid identification of human intestinal bifidobacteria by16S rRNA-targeted species-and group-species primers[J].FEMS MicrobiologyLetters, 1998, 167:113-121.
[11] 宋悦红, 宋艳文, 郭素民.几种乳酸菌的表型特征[J].职业与健康, 2006 (18) :1456-1457. [12] 陈芳, 靳亚平.奶制品中有益菌的分离鉴定[J].黑龙江畜牧兽医, 2007 (3) :102-103. [13] 徐松滨, 李威娜.酸奶中保加利亚乳杆菌的分离和鉴定[J].中国乳品工业, 2003, 31 (3) :16-18. [14] 佟潇.酸奶中嗜热链球菌的分离和鉴定[J].中国乳品工业, 2004, 32 (4) :25-27. [15] 于晓君, 时东清, 黄少云, 等.乳酸菌中双歧杆菌的检测方法探讨[J].现代测量与实验室管理, 2011 (1) :5-7. [16] Buchanan RE主编.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组译, 伯杰细菌鉴定手册[M].第八版.北京:科学出版社, 1984. [17] ISO7889-2003.Yogurt-Enumeration of characteristic microorganisms-Colony-count technique at37℃[S].
[18] GB4789.35-2010.食品安全国家标准食品微生物学检验乳酸菌检验[S]. [19] Connie S Price, Holly Huynh, Suzanne Paule, et al.Comparison of an automated ribotyping system to restriction endonuclease analysis and pulse-field gel electrophoresis for differentiating vancomycin-resistant Enterococcus faecium isolates[J].J Clin Microbiol, 2002, 40:1858-1861.
[20] Wong H C, Liu C C, Pan T M, et al.Molecular typing of Vibrio parahaemolyticus isolates, obtained from patients involved in food poisoning outbreaks in Taiwan, by random amplified polymorphic DNA analysis[J].J Clin Microbiol, 1999, 37:1809-1812.
[21] 严纪文, 马聪, 朱海明, 等.2003～2005年广东省水产品中副溶血性弧菌的主动监测及其基因指纹图谱库的建立[J].中国卫生检验杂志, 2006, 16 (4) :387-391.
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