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食品与发酵工业  2021, Vol. 47 Issue (11): 241-246    DOI: 10.13995/j.cnki.11-1802/ts.026481
  分析与检测 本期目录 | 过刊浏览 | 高级检索 |
基于数字化环介导等温扩增技术的牛乳中大肠杆菌快速精准定量分析
易昌毓1,2, 罗自生2, 潘响亮1, 林星宇2*
1(浙江工业大学 环境学院,浙江 杭州,310014)
2(浙江大学 生物系统工程与食品科学学院,浙江 杭州,310058)
Rapid detection of Escherichia coli in milk by digital loop-mediated isothermal amplification
YI Changyu1,2, LUO Zisheng2, PAN Xiangliang1, LIN Xingyu1*
1(College of Environment,Zhejiang University of Technology,Hangzhou 310014,China)
2(College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058,China)
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摘要 建立了一种数字化环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)用于牛乳中大肠杆菌的精准快速定量。通过向反应体系中加入聚乙二醇单体进行水凝胶交联,随后在65 ℃进行等温扩增。由于水凝胶基质的限制作用,一个细菌的特定基因序列只会产生一个扩增子荧光亮点,用于计数。因此,可以通过荧光显微镜进行成像并通过扩增子点数目来确定样品细菌浓度。通过对扩增时间、引物浓度、牛血清白蛋白浓度等条件进行优化,得到最优结果。结果表明,该方法具有快速、简便、高效和高灵敏性的特点,检测灵敏度可降至单个细菌,有望成为食品快速检测大肠杆菌的方法。
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易昌毓
罗自生
潘响亮
林星宇
关键词:  大肠杆菌  数字化  环介导等温扩增技术  牛乳  快速检测    
Abstract: This study aimed to establish a digital loop-mediated isothermal amplification (LAMP) method to detect Escherichia coli in milk. This experiment was performed by including two polyethylene glycol (PEG) hydrogel monomers into the LAMP assay for hydrogel cross-linking, followed by isothermal incubation at 65 ℃. Due to the restriction effect of the PEG hydrogel matrix, one bacterium could only produce one fluorescent amplicon dot for simple digital counting. Therefore, the sample bacterial concentrations can be determined based on the number of fluorescent amplicon dots using a fluorescence microscope for imaging. Optimal results were obtained by systematically optimized the experimental conditions, such as amplification time, primer concentration and bovine serum albumin concentration. The result showed that the detection could be completed with sensitivity down to single bacterium. Considering its rapidity, simplicity, efficiency and sensitivity, the developed digital LAMP has an enormous potential for rapid detection of E. coli in food samples.
Key words:  Escherichia coli    digital    loop-mediated isothermal amplification (LAMP)    milk    rapid detection
收稿日期:  2020-12-16      修回日期:  2021-02-02           出版日期:  2021-06-15      发布日期:  2021-07-16      期的出版日期:  2021-06-15
基金资助: 国家自然科学基金项目(22004107);中央高校基本科研业务费专项资金资助项目(2020QNA6007)
作者简介:  硕士研究生(林星宇研究员为通讯作者,E-mail:xingyu@zju.edu.cn)
引用本文:    
易昌毓,罗自生,潘响亮,等. 基于数字化环介导等温扩增技术的牛乳中大肠杆菌快速精准定量分析[J]. 食品与发酵工业, 2021, 47(11): 241-246.
YI Changyu,LUO Zisheng,PAN Xiangliang,et al. Rapid detection of Escherichia coli in milk by digital loop-mediated isothermal amplification[J]. Food and Fermentation Industries, 2021, 47(11): 241-246.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.026481  或          http://sf1970.cnif.cn/CN/Y2021/V47/I11/241
[1] 陈嘉惠, 陈沁,钮冰.乳品中有害微生物检测技术的研究进展[J].中国乳品工业,2019,47(5):32-36.CHEN J H,CHEN Q,NIU B.Research progress on detection techniques of harmful microorganisms in dairy products[J].China Dairy Industry,2019,47(5):32-36.
[2] 陈盼, 贺稚非,龚霄,等.食品中大肠杆菌的快速检测方法[J].食品工程,2008(1):58-61.CHEN P,HE Z F,GONG X,et al.Rapid methods for detection of Escherichia coli[J].Food Engineering,2008(1):58-61.
[3] 赵艳坤, 陈贺,王帅,等.生鲜乳中微生物检测技术研究进展[J].中国奶牛,2019(4):52-56.ZHAO Y K,CHEN H,WANG S,et al.Research status of microbial detection technology in raw milk[J].China Dairy Cattle,2019(4):52-56.
[4] 国家卫生和计划生育委员会,国家食品药品监督管理总局.GB 4789.3—2016 食品安全国家标准 食品微生物学检验 大肠菌群计数[S].北京:中国标准出版社,2016.National Health and Family Planning Commission,National Medical Products Administration.GB 4789.3-2016 National standards for food safety Microbiological inspection of food Coliform population count[S].Beijing:China Standards Press,2016.
[5] 周威, 胡梁斌,李红波,等.食物中食源性病原菌检测技术研究进展[J].食品研究与开发,2017,38(9):213-216.ZHOU W,HU L B,LI H B,et al.Advances in detection of foodborne pathogens in food[J].Food Research and Development,2017,38(9):213-216.
[6] 马冬. 乳品卫生指标大肠杆菌及检测技术的研究[D].西安:陕西科技大学,2009.MA D.Study on important sanitation indexes and detecting technology of E.coli in milk[D].Xi'an:Shaanxi University of Science & Technology,2009.
[7] 方婷子, 史贤明,施春雷.沙门氏菌血清型快速PCR鉴定方法的建立[J].中国食品学报,2017,17(2):212-219.FANG T Z,SHI X M,SHI C L.Development of rapid PCR determination method of Salmonella serovars[J].Journal of Chinese Institute of Food Science and Technology,2017,17(2):212-219.
[8] 杜雄伟,李叶,冮洁,等.肉制品中沙门氏菌invA基因实时荧光定量PCR检测方法的建立[J].食品工业科技,2013,34(12):68-70;80.DU X W,LI Y,GANG J,et al.Establishment of real-time fluorescent PCR detection method of invA gene in Salmonella[J].Science and Technology of Food Industry,2013,34(12):68-70;80.
[9] HEYRIES K A,TROPINI C,VANINSBERGHE M,et al.Megapixel digital PCR[J].Nature Methods,2011,8(8):649-651.
[10] LIN X Y,HUANG X,ZHU Y Z,et al.Asymmetric membrane for digital detection of single bacteria in milliliters of complex water samples[J].ACS Nano,2018,12(10):10 281-10 290.
[11] NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12).DOI:10.1093/nar/28.12.e63.
[12] 郭建平,万佳佳,陆兆新,等.基于可视化环介导等温扩增技术快速检测金黄色葡萄球菌[J].食品科学,2019,40(20):325-331.GUO J P,WAN J J,LU Z X,et al.Rapid visual detection of Staphylococcus aureus using loop-mediated isothermal amplification[J].Food Science,2019,40(20):325-331.
[13] SONG H,BAE Y C,KWON H,et al.Loop-mediated isothermal amplification assays for Enterococcus sp.,Escherichia coli and Staphylococcus aureus in chicken[J].FEMS Microbiology Letters,2019,366(5).DOI:10.1093/femsle/fnz042.
[14] 申培立, 王羽,李志辉,等.变形杆菌环介导等温扩增检测法的可视化方法的比较[J].河北农业大学学报,2016,39(9):73-77;82.SHEN P L,WANG Y,LI Z H,et al.Comparison of the visualization methods of loop-mediated isothermal amplification for detecting Proteus[J].Journal of Agricultural University of Hebei,2016,39(9):73-77;82.
[15] 王瑾, 林丽萍,郜彦彦,等.实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌[J].食品科学,2016,37(24):170-174.WANG J,LIN L P,GAO Y Y,et al.Development and application of a real-time loop-mediated isothermal amplification assay for rapid detection of Salmonella enterica ser.enteritis retrieved from chicken[J].Food Science,2016,37(24):170-174.
[16] 魏桢元. 环介导等温扩增技术在食源性沙门氏菌检测中的应用研究进展[J].江苏农业科学,2020,48(3):80-85.WEI Z Y.Research progress on application of loop-mediated isothermal amplification (LAMP) for detection of Salmonella in food[J].Jiangsu Agricultural Sciences,2020,48(3):80-85.
[17] YUAN H,CHAO Y C,SHUM H C.Droplet and microchamber-based digital loop-mediated isothermal amplification (dLAMP)[J].Small,2020,16.DOI:10.1002/smll.201904469.
[18] MITRA R D,CHURCH G M.In situ localized amplification and contact replication of many individual DNA molecules[J].Nucleic Acids Research,1999,27(24),e34-e39.
[19] HILL J,BERIWAL S,CHANDRA I,et al.Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli[J].Journal of Clinical Microbiology,2008,46(8):2 800-2 804.
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[2] LI Xiao-min et a. The influence of feruloyl esterase on filterability of wort[J]. Food and Fermentation Industries, 2017, 43(11): 30 -33 .
[3] LIU Wen-ying et al. Protective effect of wheat oligopeptides and glutamine against gastrointestinal mucosa damage in rats[J]. Food and Fermentation Industries, 2017, 43(11): 51 -57 .
[4] LI Bin et al.

Vacuum drying kinetics characteristics of Chinese prickly ash based on Weibull distribution [J]. Food and Fermentation Industries, 2017, 43(11): 58 -64 .

[5] TIAN Jun-qing et al. Synthesis and structure characterization of octenyl succinic acid cellulose ester #br#[J]. Food and Fermentation Industries, 2017, 43(11): 65 .
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[7] QIN Ya-nan et a. Study on oxidative stability of grape seed oil[J]. Food and Fermentation Industries, 2017, 43(11): 95 .
[8] ZHANG Yang et al. Effects of Material Composition on the Oil Absorption of Restructured Potato Chips[J]. Food and Fermentation Industries, 2017, 43(11): 140 .
[9] LI Sheng et al. Effects of three improvers on the quality of dried noodles with high content of purple sweet potato flour[J]. Food and Fermentation Industries, 2017, 43(11): 146 .
[10] TIAN Xue-zhi et al. Effect of high hydrostatic pressure treatment on the functional properties of konjac glucomannan / soybean protein composite sol[J]. Food and Fermentation Industries, 2017, 43(11): 153 .
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