pH响应比色酶联免疫吸附法检测牛奶中猪霍乱沙门氏菌

李倩影; 张抗抗; 周耀锋; 黄小林; 李响敏; 熊勇华

doi:10.13386/j.issn1002-0306.2019100100

pH响应比色酶联免疫吸附法检测牛奶中猪霍乱沙门氏菌

李倩影^1,2,
张抗抗^1,2,
周耀锋^1,2,
黄小林^1,2,
李响敏^1,2, ,,
熊勇华^1,2

1. 食品科学与技术国家重点实验室, 江西南昌 330047;
2. 南昌大学食品学院, 江西南昌 330031

基金项目:

"十三五"国家重点研发计划（2018YFC1602202、2018YFC1602203、2018YFC1602505）；国家自然科学基金（31760485、31901780）。

详细信息

作者简介:
李倩影(2000-),女,学士,研究方向:食品安全快速检测,E-mail:1464561886@qq.com;张抗抗(1998-),女,学士,研究方向:食品安全快速检测,E-mail:m15797899516@163.com。

通讯作者:
李响敏(1973-),男,博士,讲师,研究方向:食品质量与控制,E-mail:lixiangmin73@163.com。

中图分类号: TS207.4
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出版历程
- 收稿日期: 2019-10-15
- 网络出版日期: 2021-01-07
- 刊出日期: 2020-12-31

pH Responded Colorimetric ELISA for Sensitive Detection of Salmonella Choleraesuis in Milk

1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China;
2. School of Food Science and Technology, Nanchang University, Nanchang 330031, China

摘要

摘要: 人类常常因误食被猪霍乱沙门氏菌感染的食物而引发中毒。因此，急需构建适用于快速、灵敏检测食物中猪霍乱沙门氏菌污染的新方法。鉴于此，本文构建了一种pH响应比色酶联免疫吸附法灵敏检测牛奶中猪霍乱沙门氏菌。该方法利用葡萄糖氧化酶催化底物葡萄糖产生葡萄糖酸，导致溶液pH下降，进而引发溴甲酚紫（Bromocresol purple，BCP）溶液颜色由紫色变成亮黄色，随后通过记录BCP颜色变化（OD₄₃₀/OD₅₉₀）实现目标菌的定量检测。当菌浓度为2.54×10³~6.17×10⁵ CFU/mL，该方法定量检测猪霍乱沙门氏菌可用方程一表述：y₁=0.1051ln （x）+0.7024（R²=0.7513）；当菌浓度为6.17×10⁵~1.67×10⁷ CFU/mL，该方法定量检测猪霍乱沙门氏菌可用方程二表述：y₂=1.3216ln （x）-15.797（R²=0.9711）；当菌浓度大于1.67×10⁷ CFU/mL时，BCP溶液呈现明显亮黄色，OD₄₃₀/OD₅₉₀值趋于稳定，无法实现猪霍乱沙门氏菌定量检测。将六个不同浓度的猪霍乱沙门氏菌（2.5×10³~5.6×10⁶ CFU/mL）加标至牛奶中，检测结果显示回收率介于72.16%~103.58%，相对标准偏差介于7.54%~15.30%。总之，本研究所构建的比色ELISA方法适用于牛奶中不同浓度的猪霍乱沙门氏菌快速、灵敏定量检测。
- pH响应比色酶联免疫吸附法(pH-ELISA) /
- 葡萄糖氧化酶 /
- 溴甲酚紫 /
- 猪霍乱沙门氏菌
Abstract: Human maybe get poisoning after eating Salmonella choleraesuis-infected food. Thus,developing a new analytical technology for rapid and sensitive detection of Salmonella choleraesuis in food is urgently needed. Hence,a pH responded colorimetric enzyme-linked immunosorbent assay(pH-ELISA)was developed for sensitive detection of Salmonella choleraesuis in milk. In this method,glucose oxidase(GOx)was used to catalyze glucose for the generation of gluconic acid. The color of bromocresol purple(BCP)was changed from purple to bright yellow because the pH of sample solution decreased. Under the optimal conditions,the proposed method achieved two linear independent regression equations. When Salmonella choleraesuis concentration increased from 2.54×10³ to 6.17×10⁵ CFU/mL,the first regression equation was described as y₁=0.1051ln(x)+0.7024(R²=0.7513). With the increase of Salmonella choleraesuis concentration from 6.17×10⁵ to 1.67×10⁷ CFU/mL,the second regression equation was expressed as y₂=1.3216ln(x)-15.797(R²=0.9711). The average recoveries of the proposed method for spiked milk samples at Salmonella choleraesuis concentrations ranging from 2.5×10³~5.6×10⁶ CFU/mL were 72.16%~103.58% with the relative standard deviations ranging from 7.54%~15.30%. In conclusion,the developed colorimetric ELISA is suitable for the rapid and sensitive quantitation of Salmonella choleraesuis in milk.
- pH responded colorimetric enzyme-linked immunosorbent assay(pH-ELISA) /
- glucose oxidase /
- bromocresol purple /
- Salmonella choleraesuis

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参考文献(26)

[1]	Kordas A,Papadakis G,Milioni D,et al. Rapid Salmonella detection using an acoustic wave device combined with the RCA isothermal DNA amplification method[J]. Sensing and Bio-Sensing Research,2016,11(2):121-127.
[2]	Wang S,Zheng L,Cai G,et al. A microfluidic biosensor for online and sensitive detection of Salmonella typhimurium using fluorescence labeling and smartphone video processing[J]. Biosensors and Bioelectronics,2019,140:111333.
[3]	郭耀东,韩晓江,张英华,等. 沙门氏菌检测的研究进展[J]. 农产品加工,2019(9):75-78.
[4]	王毳,闫磊,曾庆祝. 沙门氏菌的检测技术与方法[J]. 现代食品科技,2007(5):82-85,75.
[5]	Chen J,Zhang L,Paoli G C,et al. A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis[J]. International Journal of Food Microbiology,2009,137(2):168-174.
[6]	Grant A Q,Hashem F,Parveen S. Salmonella and campylobacter:Antimicrobial resistance and bacteriophage control in poultry[J]. Food Microbiology,2016,53:104-109.
[7]	彭亚博,李晓婷,方婷,等. 鸡肉中沙门氏菌和背景菌群动力学模型的研究[J]. 食品科学,2019(9):7-15.
[8]	Hu L,Ma L M,Zheng S,et al. Development of a novel loop-mediated isothermal amplification(LAMP)assay for the detection of Salmonella ser. Enteritidis from egg products[J]. Food Control,2018,88:190-197.
[9]	Iñiguez-moreno M,Gutiérrez-lomelí M,Avila-novoa M G. Kinetics of biofilm formation by pathogenic and spoilage microorganisms under conditions that mimic the poultry,meat,and egg processing industries[J]. International Journal of Food Microbiology,2019,303:32-41.
[10]	朱海华,章建军,尹红娜,等. 一种快速富集生乳中沙门氏菌的方法[J]. 食品科技,2018,43(2):302-306.
[11]	Pinto M S,Pires A C S,Sant'ana h M P,et al. Influence of multilayer packaging and microfiltration process on milk shelf life[J]. Food Packaging and Shelf Life,2014,1(2):151-159.
[12]	梁攀,董萍,王洋,等. 免疫学技术在食品安全快速检测中的应用研究进展[J]. 食品安全质量检测学报,2018,9(9):2085-2089.
[13]	马兰,王淑娟,曾海娟,等. 侧流层析技术研究进展[J]. 食品科学,2018,39(15):333-342.
[14]	张帅,齐颖颖,张红星,等. 双抗夹心酶联免疫吸附快速检测冷鲜肉中的6种血清型沙门氏菌[J]. 食品科学,2016,37(16):211-215.
[15]	Chen R,Huang X,Li J,et al. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots[J]. Analytica Chimica Acta,2016,947:50-57.
[16]	Wang C,Xing K,Zhang G,et al. Novel ELISA based on fluorescent quenching of DNA-stabilized silver nanoclusters for detecting E. coli O157:H7[J]. Food Chemistry,2019,281:91-96.
[17]	Gao B,Chen X,Huang X,et al. Urease-induced metallization of gold nanorods for the sensitive detection of Salmonella enterica Choleraesuis through colorimetric ELISA[J]. Journal of Dairy Science,2019,102(3):1997-2007.
[18]	Hu J,Huang R,Sun Y,et al. Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks[J]. Journal of Microbiological Methods,2019,158:25-32.
[19]	黄震,夏诗琪,刘道峰,等. 基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌[J]. 分析化学,2017,45(2):217-223.
[20]	夏诗琪,徐超莲,刘道峰,等. 胶体金免疫层析法联检食品中5种典型沙门氏菌模型的建立和优化[J]. 食品科学,2014,35(22):154-158.
[21]	伍燕华,牛瑞江,赖卫华,等. 双抗夹心酶联免疫吸附法检测沙门氏菌[J]. 食品工业科技,2014,35(10):62-65.
[22]	Lvov Y,Antipov A A,Mamedov A,et al. Urease encapsulation in nanoorganized microshells[J]. Nano Letters,2001,1(3):125-128.
[23]	Xiong Y,Pei K,Wu Y,et al. Colorimetric ELISA based on glucose oxidase-regulated the color of acid-base indicator for sensitive detection of aflatoxin B₁in corn samples[J]. Food Control,2017,78:317-323.
[24]	中华人民共和国卫生部. GB 4789.4-2016食品安全国家标准食品微生物学检验沙门氏菌检验[S]. 北京:中国标准出版社,2016:1-7.
[25]	Gokduman K,Avsaroglu M D,Cakiris A,et al. Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples[J]. Journal of Microbiological Methods,2016,122:50-58.
[26]	刘立兵,耿云云,姜彦芬,等. 沙门氏菌重组酶聚合酶检测方法的建立及应用[J]. 食品科学,2019,40(2):298-303.

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