Microsoft Word _排版二校出4581

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1 7 11 Vol. 7 No Journal of Food Safety and Quality Nov., 2016 张蕴哲, 张先舟, 李英军, 马晓燕 *, 张伟 (, ) 摘要 : 目的 (loop-mediated isothermal amplification, LAMP) 方法 16S rrna, LAMP,, 结果, 5.5 h, 62 CFU/mL, 120 CFU/mL, % 结论, 关键词 : ; ; Rapid detection of Lactobacillus acidophilus in yogurt by loop-mediated isothermal amplification ZHANG Yun-Zhe, ZHANG Xian-Zhou, LI Ying-Jun, MA Xiao-Yan *, Zhang Wei (College of Food Science & Technology, Hebei Agriculture University, Baoding , China) ABSTRACT: Objective To establish a method for detection of thermophilic Lactobacillus in yogurt by loop mediated isothermal amplification (LAMP). Methods Using the 16S rrna of Lactobacillus acidophilus as target sequences, the primers were designed and the LAMP reaction system and produce were built and optimized. At the same time, the specificity of primers was verified and the sensitivity and detection limit of Lactobacillus acidophilus were confirmed. Results The reaction results were determined by ladder strip through gel electrophoresis and white precipitate of magnesium pyrophosphate after centrifugation. Detection time was 5.5 h. The sensitivity and detection limit by artificial contamination were 62 CFU/mL and 120 CFU/mL, respectively. The specific results of 12 strains of Lactobacillus acidophilus bacteria were 100%, which showed the primers had high specificity. Conclusion LAMP can be used to detect Lactobacillus acidophilus in yogurt with high sensitivity, high specificity, simple operation and short time consuming. KEY WORDS: loop-mediated isothermal amplification; Lactobacillus acidophilus; yogurt 1 引言 (Lactobacillus acidophilus), 3,,, 基金项目 : (C ) ; (QN ) Fund: Supported by the National Natural Science Foundation of China (C ); Suppotede by the Youth Foundation of of Higher Education of Hebei (QN ) * 通讯作者 :,,, wjxmxy@163.com *Corresponding author: MA Xiao-Yan, Associate Professor, Master Tutor, College of Food Science & Technology, Hebei Agriculture University, Baoding , China. wjxmxy@163.com

2 [1,2], [3-5],, [6],,,, [7,8] PCR(quantitative real-time PCR, qpcr),, [9-12] (loop-mediated isothermal amplification, LAMP) 2000 [13] 4 1 BstDNA, 60~65, DNA 10 9, 2,, LAMP [14-16], LAMP LAMP 16S rrna, 2 材料与方法 2.1 材料试验菌株 (Lactobacillus acidophilus, NCFM, (China's Industrial Microbial Preservation Management Center, CICC) 22150); (Lactobacillus bulgaricus LB01, ); (Lactobacillus bulgaricus 5M1, ); (Lactobacillus bulgaricus 5M2, ); (Streptococcus thermophilus St001, ); (Shigella sonnei, CMCC 51334); (Escherichia coli, CMCC 44102); (Proteus vulgaris, CMCC 49027); (Enterobacter sakazakii, (American Type Culture Collection, ATCC) 29544); (Salmonella enteritidis, CMCC 50041); (Salmonella schottmuelleri, CMCC 50001); (Bacilus cereus, CMCC 66302); (Clostridium perfringens, CMCC 64609) 样品主要培养基和试剂 MRS MRS ( ); Bst DNA (New England Biolab ); dntps MgCl 2 DNAMarker DNA ( ( ) )); (, ) 仪器与设备 Whatman T Gradient PCR ( Biometra ); ( ); DXY-33A ( ); UVIpro ( ); TGL-16B ( ); SPX-150B-Z ( ); DY-H21 ( ); LS-30 ( ); 2.2 试验方法菌种的培养 : MRS 2, MRS, 37, : 2,, 37, 细菌基因组 DNA 的提取, LAMP 反应引物的设计 GenBank 16S rrna, ( jp/lamp3.0.0/index.html), 1 表 1 LAMP 引物 Table 1 Primers of LAMP (5' 3') F 3 AGCGGTGGAATGCGTAGA B 3 FIP BIP AGTGCTTAATGCGTAGTGCA TCGAGCCTCAGCGTCAGTTG-TATGGAA GAACACCAGTGGC TTGATACCCTGGTGTCCATGCC-TGAGA GGCGGAAACCTCC LF CAGACCAGAGAGCCGCCTTC LB TAAACGATGAGTGCTAGTGTT LAMP 反应体系的建立, 25 μl : Bst DNA 10 U 10 Bst DNA 2 μl FIP BIP 1.8 μmol/l F 3 B μmol/l LF LB 0.9 μmol/l dntp 5 mmol/l MgSO 4 2 mmol/l

11, : 4583 DNA 1 μl, 25 μl : 63 20 min, 80 10 min : 5 μl 1.5%,, 2.2.5 LAMP 引物特异性的检测 12 DNA,, LAMP, 5 μl 1.

2.6 嗜酸乳杆菌的灵敏度检测 MRS, 37 12 h, 10, 1 ml DNA, LAMP, 5 μl 1.5% 2.2.7 酸奶中嗜酸乳杆菌的检出限

12000 r/min 1 min,, 1 ml ; DNA,, LAMP, 5 μl 1.5% 2, LAMP 16S rrna 1 LAMP Fig.

1.2 LAMP 扩增产物的电泳分析 LAMP 2, 2, DNA 1 LAMP 2 LAMP Fig.

3 11, : 4583 DNA 1 μl, 25 μl : min, min : 5 μl 1.5%,, LAMP 引物特异性的检测 12 DNA,, LAMP, 5 μl 1.5%, LAMP 嗜酸乳杆菌的灵敏度检测 MRS, h, 10, 1 ml DNA, LAMP, 5 μl 1.5% 酸奶中嗜酸乳杆菌的检出限 MRS, h; 10%, min,, 4%,, 37 4 h, 4 10 h; 25 ml, 10, MRS ; 10 ml, r/min 1 min,, 1 ml ; DNA,, LAMP, 5 μl 1.5% 2, LAMP 16S rrna 1 LAMP Fig. 1 The visualization result of LAMP 1- ; 2-3 结果与分析 3.1 LAMP 反应体系的建立 LAMP 扩增产物的眼观分析 LAMP 1, 1, DNA 2, 1, LAMP 16S rrna LAMP 扩增产物的电泳分析 LAMP 2, 2, DNA 1 LAMP 2 LAMP Fig. 2 The electrophoresis result of LAMP M-100 bp ladder marker; 1- ; 引物特异性的研究 12 DNA, LAMP, LAMP 3, LAMP,, LAMP LAMP

4584 3.3 食品安全质量检测学报第7卷 LAMP 检测嗜酸乳杆菌的灵敏度带, 即 LAMP 检测嗜酸乳杆菌的灵敏度为 62 CFU/mL 培养 12 h 的嗜酸乳杆菌 MRS 肉汤培养物, 经平板菌 3.4 8 LAMP 检测酸奶中嗜酸乳杆菌的检出限落计数测得菌液浓度为 6.

2 107 稀释, 每个稀释度取 1 ml, 按试剂盒法提取基因组 DNA CFU/mL 每个稀释度经处理后按试剂盒法提取基因组作为模板进行 LAMP 反应, 扩增产物进行电泳分析 DNA 作为模板, 进行 LAMP 反应从图 5 中可看出, 稀释 LAMP

3 The specificity test of LAMP primers M-100 bp ladder marker; 0-阴性对照; 1-CMCC 51334; 2-CMCC 44102; 3-CMCC 49027; 4-ATCC 29544; 5-CMCC 50041; 6-CMCC 50001;

4 The sensitivity of Lactobacillus acidophilus by LAMP M-100 bp ladder marker; 1-阳性对照; 2-阴性对照; 3-6.2 108 CFU/mL; 4-6.2 107 CFU/mL; 5-6.2 106 CFU/mL; 6-6.

4 食品安全质量检测学报第7卷 LAMP 检测嗜酸乳杆菌的灵敏度带, 即 LAMP 检测嗜酸乳杆菌的灵敏度为 62 CFU/mL 培养 12 h 的嗜酸乳杆菌 MRS 肉汤培养物, 经平板菌 LAMP 检测酸奶中嗜酸乳杆菌的检出限落计数测得菌液浓度为 CFU/mL, 进行 10 倍梯度酸奶样品中嗜酸乳杆菌的初始浓度为稀释, 每个稀释度取 1 ml, 按试剂盒法提取基因组 DNA CFU/mL 每个稀释度经处理后按试剂盒法提取基因组作为模板进行 LAMP 反应, 扩增产物进行电泳分析 DNA 作为模板, 进行 LAMP 反应从图 5 中可看出, 稀释 LAMP 检测嗜酸乳杆菌的灵敏度结果见图 4, 由图 4 可知, 倍数达到 106(12 CFU/mL)时, 电泳图中不再出现梯形条带, 8 0 当稀释倍数达到 10 ( CFU/mL)时不再出现梯形条即 LAMP 检测酸奶中嗜酸乳杆菌的检出限为 120 CFU/mL 图 3 LAMP 引物特异性的检测结果 Fig. 3 The specificity test of LAMP primers M-100 bp ladder marker; 0-阴性对照; 1-CMCC 51334; 2-CMCC 44102; 3-CMCC 49027; 4-ATCC 29544; 5-CMCC 50041; 6-CMCC 50001; 7-CMCC 66302; 8-CMCC 64609; 9-St001; 10-5M2; 11-5M1;12-LB01; 13-CICC 图 4 LAMP 检测嗜酸乳杆菌的灵敏度 Fig. 4 The sensitivity of Lactobacillus acidophilus by LAMP M-100 bp ladder marker; 1-阳性对照; 2-阴性对照; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL 图 5 LAMP 检测酸奶中嗜酸乳杆菌的检出限 Fig. 5 Detection limit of Lactobacillus acidophilus in yoghurt products by LAMP M-100 bp ladder marker; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; CFU/mL; 8-阳性对照;

11, : 4585 4 结论,, 16S rrna,, LAMP,,, LAMP MRS,, 48 h,, LAMP 5.5 h DNA LAMP, ; PCR,,,, DNA, DNA, LAMP, 参考文献 [1],,. [J]., 2011, 23(3): 43 46. Li J, Li BR, Li Q.

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid [J]. Proc Natl Acad Sci USA, 2011, 108(Suppl 1): 4623 4630. [3],. [J]., 2009, 22(12): 79 80.

5 11, : 结论,, 16S rrna,, LAMP,,, LAMP MRS,, 48 h,, LAMP 5.5 h DNA LAMP, ; PCR,,,, DNA, DNA, LAMP, 参考文献 [1],,. [J]., 2011, 23(3): Li J, Li BR, Li Q. Lactobacillus acidophilus dairy probiotic properties [J]. J Heilongjiang Bayi Agric Univ, 2011, 23(3): [2] Mohamadzadeh M, Pfeiler EA, Brown JB, et al. Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid [J]. Proc Natl Acad Sci USA, 2011, 108(Suppl 1): [3],. [J]., 2009, 22(12): Li Q, Lu N. Research on the actuality and trend of Lactobacillus acidophilus [J]. Sci Technol Entrepreneurship Monthly, 2009, 22(12): [4],. [J]., 2008, 44(9): Guo X, Luo XY. Lactobacillus acidophilus and research progress in applying to medicine [J]. Inner Mongolia Med J, 2008, 44(9): [5],. [J]., 2015, (9): Zhong HX, Qi SS. Research progress of Lactobacillus acidophilus [J]. Heilongjiang Anim Sci Vet Med, 2015, (9): [6],. [J]., 2008, 31(l): Ying J, Zheng XP. Enumeration of Lactobacillus acidophilus from yoghurt [J]. Dairy Sci Technol, 2008, 31(l): [7]. [J]., 2011, 34(3): Jiang SG. Study on test methods for Lactobacillus acidophilus in yogurt [J]. Dairy Sci Technol, 2011, 34(3): [8] Gomes AA, Braga SP, Cruz AG, et al. Effect of the inoculation level of Lactobacillus acidophilus, in probiotic cheese on the physicochemical features and sensory performance compared with commercial cheeses [J]. J Dairy Sci, 2011, 94(10): [9],,,. RT-PCR [J]., 2014, 23(7): Kuo ZH, Fang H, Xia ZS, et al. Application of quantitative RT-PCR detection in fermentation materials of Lactobacillus acidophilus cell number [J]. Feed Res, 2014, 23(7): [10] Sheu SJ, Hwang WZ, Chen HC, et al. Development and use of tuf gene-based primers for the multiplex PCR detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum in commercial dairy products [J]. J Food Protect, 2009, 72(1): [11] Berger B, Pridmore RD, Barretto C, et al. Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics [J]. J Bacteriol, 2007, 189(4): [12],,,. [J]., 2012, 33(11): Bao QH, Li MH, Xu J, et al. The qualitative and quantitative method for detecting Lactobacillus acidophilus in fermented milk [J]. Biotechnology, 2012, 33(11): [13] Nimitphak T, Kiatpathomchai W, Flegel TW. Loop-mediated isothermal amplification of DNA [J]. Nucleic Acids Res, 2000, 28(12): E63 E63. [14],. [J]., 2016, 7(3): Fan YL, Yang MC. Rapid detection of Bifidobacterium spp. by loopmediated isothermal amplification [J]. J Food Saf Qual, 2016, 7(3): [15] Lloyd A, Pasupuleti V, Thota P, et al. Accuracy of loop-mediated isothermal amplification for the diagnosis of Clostridium difficile, infection: a systematic review [J]. Diagn Microbiol Infect Dis, 2015, in press(1): [16],,. [J]., 2016, 32(5): Hu ZY, Xu ZH, Lu YY. Analysis of common issues research progress in loop mediated isothermal amplification [J]. Chin J Virol, 2016, 32(5): 作者简介 ( 责任编辑 : 姚菲 ) 张蕴哲, 学士, 研究实习员, 主要研究方向为微生物检验 mickey @126.com 马晓燕, 副教授, 硕士生导师, 主要研究方向为食品加工与食品安全 wjxmxy@163.com

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<4D6963726F736F667420576F7264202D203631372D3632312032303133303430333030312DBACEC0F25FD0A3B6D4B8E55F2DB6FED0A32D2D2DC8A5B5F4CDBCD6D0B5C4BBD8B3B5B7FBBAC52E646F63> 第 4 卷第 2 期食品安全质量检测学报 Vol. 4 No. 2 2013 年 4 月 Journal of Food Safety and Quality Apr., 2013 何莉 *, 姜笑寒 ( 广东省食品药品职业技术学校, 广州 510663) 摘要 : 本文通过科学地运用 html5+jsp+sql 技术, 建

Microsoft Word _排版 二校 出4581

Microsoft Word _排版二校出4581