微流控芯片恒温扩增技术快速检测米饭中的蜡样芽孢杆菌

doi:10.3785/j.issn.1008-9209.2020.06.101

浙江大学学报（农业与生命科学版）

2021, Vol. 47

Issue (2): 193-202 DOI: 10.3785/j.issn.1008-9209.2020.06.101

食品科学

微流控芯片恒温扩增技术快速检测米饭中的蜡样芽孢杆菌

王珍¹(

),贺磊²,肖英平³,卢先东²,刘艳红²,陆雯¹,王首锋⁴(

)

^1.绿城农科检测技术有限公司，杭州 310052
^2.宁波爱基因科技有限公司，浙江宁波 315000
^3.浙江省农业科学院，杭州 310021
^4.浙江大学医学院基础医学系，杭州 310058

Rapid detection of Bacillus cereus in rice by isothermal amplification with microfluidic chip method

Zhen WANG¹(

),Lei HE²,Yingping XIAO³,Xiandong LU²,Yanhong LIU²,Wen LU¹,Shoufeng WANG⁴(

)

^1.Greentown Agricultural Testing Technology Co. , Ltd. , Hangzhou 310052, China
^2.Ningbo iGene Technology Co. , Ltd. , Ningbo 315000, Zhejiang, China
^3.Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
^4.Department of Basic Medicine, School of Medicine, Zhejiang University, Hangzhou 310058, China

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摘要：

为建立米饭中的蜡样芽孢杆菌微流控芯片恒温扩增快速检测方法，利用蜡样芽孢杆菌公开的hblA基因序列设计引物，加入荧光染料SYTO-9，采用微流控芯片通过环介导恒温扩增进行实时荧光读数，并通过24株菌株验证其特异性；分别用阳性质粒和阳性菌株测试其灵敏度、检出限和重复性。结果表明：2株蜡样芽孢杆菌呈阳性，22株非蜡样芽孢杆菌呈阴性；微流控芯片恒温扩增技术检测蜡样芽孢杆菌菌液的灵敏度为170 CFU/mL，检测时间在35 min内；使用合成的蜡样芽孢杆菌阳性质粒样品，其灵敏度为10 μL^－1，检测时间在15 min内，比传统分离鉴定方法的灵敏度高10倍；人工污染米饭中蜡样芽孢杆菌的检出限为570 CFU/g，并可在45 min内完成结果判定；蜡样芽孢杆菌阳性质粒检测重复性好，其变异系数（coefficient of variation, CV）为2.02%。综上所述，微流控芯片恒温扩增快速检测方法特异性好、灵敏度高、结果准确，可用于米饭类食品中蜡样芽孢杆菌的快速检测。

关键词： 微流控芯片; 环介导恒温扩增; 蜡样芽孢杆菌; 米饭; 检测

Abstract:

Rapid detection of Bacillus cereus in rice by loop-mediated isothermal amplification (LAMP) with microfluidic chip method was established. Based on the published B. cereus hblA gene sequence, primers were designed, and the fluorescent dye SYTO-9 was added to the reaction system, which allowed to be tested in real-time fluorescence reading performed by LAMP with microfluidic chip. The specificity was verified by 24 strains, and the sensitivity, limit of detection (LOD) and repeatability were tested by positive plasmid and positive strain of B. cereus, respectively. The results showed that two strains of B. cereus were positive and 22 strains of non-B. cereus were negative. The sensitivity for B. cereus was 170 CFU/mL for pure cultures in 35 min and 10 μL^－1 for positive plasmids in 15 min. The sensitivity of this method was 10 times higher than that of the traditional method. The LOD for B. cereus inartificially contaminated rice was 570 CFU/g, and could be completed in 45 min. The detecting repeatability of the positive plasmid from B. cereus was good and its coefficient of variation (CV ) was 2.02%. It is indicated that LAMP with microfluidic chip method has high specificity, sensitivity and accuracy. It could be used for rapid detection of B. cereus in rice.

Key words: microfluidic chip loop-mediated isothermal amplification (LAMP) Bacillus cereus rice detection

收稿日期: 2020-06-10 出版日期: 2021-04-25

CLC:

Q 78

基金资助: 国家重点研发计划(2018YFC1603400)

通讯作者: 王首锋 E-mail: 705878750@ qq.com;sfwang@zju.edu.cn

作者简介: 王珍（https://orcid.org/0000-0001-6002-4272），E-mail：705878750@ qq.com

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引用本文:

王珍,贺磊,肖英平,卢先东,刘艳红,陆雯,王首锋. 微流控芯片恒温扩增技术快速检测米饭中的蜡样芽孢杆菌[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(2): 193-202.

Zhen WANG,Lei HE,Yingping XIAO,Xiandong LU,Yanhong LIU,Wen LU,Shoufeng WANG. Rapid detection of Bacillus cereus in rice by isothermal amplification with microfluidic chip method. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(2): 193-202.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.06.101 或 http://www.zjujournals.com/agr/CN/Y2021/V47/I2/193

表1 实验用菌株

表2 引物序列

图1 LAMP反应温度优化结果

图2 蜡样芽孢杆菌微流控芯片LAMP特异性测试结果图A中，1：蜡样芽孢杆菌[CMCC（B） 63301]；2：苏云金芽孢杆菌（ATCC 10792）；3：蕈状芽孢杆菌（ATCC 10206）；4：枯草芽孢杆菌[CMCC （B） 63501]；5：肺炎克雷伯氏菌（ATCC 4352）；6：大肠埃希菌（ATCC 25922）；7：肠出血性大肠埃希菌O157∶H7（CICC 21530）；8：阴性对照。图B中，1：蜡样芽孢杆菌[CMCC（B） 63301]；2：鼠伤寒沙门菌[CMCC（B） 50115]；3：阪崎肠杆菌（ATCC 29544）；4：马红球菌（ATCC 6939）；5：副溶血性弧菌（ATCC 17802）；6：金黄色葡萄球菌（ATCC 6538）；7：表皮葡萄球菌[CMCC（B） 26069]；8：阴性对照。图C中，1：蜡样芽孢杆菌（LCLY 001）；2：粪肠球菌（ATCC 29212）；3：单核细胞增生李斯特氏菌（ATCC 19115）；4：伊氏李斯特氏菌（ATCC 19119）；5：铜绿假单胞菌（ATCC 27853）；6：福氏志贺氏菌[CMCC（B） 51572]；7：乙型溶血性链球菌[CMCC （B） 32210]；8：阴性对照。图D中，1：蜡样芽孢杆菌[CMCC（B） 63301]；2：短小芽孢杆菌[CMCC（B） 63202]；3：地衣芽孢杆菌[CMCC（B） 63519]；4：解淀粉芽孢杆菌（ATCC 23845）；5：巨大芽孢杆菌（ATCC 35985）；6～8：阴性对照。

图3 蜡样芽孢杆菌实际样品微流控芯片LAMP反应灵敏度检测结果1～5：分别为1.7×105、1.7×104、1.7×103、1.7×102、1.7×101 CFU/mL的培养物；6：阴性对照。

图4 阳性质粒pUC57-hblA微流控芯片LAMP反应灵敏度检测结果1：阴性对照；2～8：分别为1×100、1×101、1×102、1×103、1×104、1×105、1×106 μL－1的质粒样品。

图5 阳性质粒pUC57-hblA微流控芯片LAMP的重复性检测结果1～8：1×104 μL－1的质粒重复检测8次的结果。

表3 微流控芯片LAMP检测蜡样芽孢杆菌的变异系数分析

图6 微流控芯片LAMP检测人工污染米饭中蜡样芽孢杆菌的检出限1～7：菌液浓度分别为5.7×106、5.7×105、5.7×104、5.7×103、5.7×102、5.7×101、5.7×100 CFU/mL的米饭样品；8：阴性对照。

图7 微流控芯片

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