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10μL-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。
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 realtime 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 10μL-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 in artificially 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.
作者
王珍
贺磊
肖英平
卢先东
刘艳红
陆雯
王首锋
WANG Zhen;HE Lei;XIAO Yingping;LU Xiandong;LIU Yanhong;LU Wen;WANG Shoufeng(Greentown Agricultural Testing Technology Co.,Ltd.,Hangzhou 310052,China;Ningbo iGene Technology Co.,Ltd.,Ningbo 315000,Zhejiang,China;Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Department of Basic Medicine,School of Medicine,Zhejiang University,Hangzhou 310058,China)
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2021年第2期193-202,共10页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
国家重点研发计划(2018YFC1603400)。
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
microfluidic chip
loop-mediated isothermal amplification(LAMP)
Bacillus cereus
rice
detection
