摘要
为建立一种快速、准确同时检测5种致病性弧菌的高通量液相芯片技术,本研究针对霍乱弧菌、副溶血弧菌、创伤弧菌、溶藻弧菌和拟态弧菌的10个目的基因,包括5个种特异性基因和5个毒力基因,分别设计特异性引物和探针,多重PCR扩增后的产物与偶联有不同核酸探针的微球混合物进行杂交反应,利用液相芯片检测仪分析结果。结果显示,本方法同时检测这5种致病性弧菌时,检出限最高达到1.8×10^(2)CFU/mL;检测包括大肠杆菌、金黄色葡萄球菌、河流弧菌、沙门菌、李斯特杆菌和铜绿杆菌等常见细菌时,均无交叉反应,表明特异性好;检测不同批次的样品时,变异系数均小于8%,表明重复性良好。本研究建立的液相芯片检测方法能够快速同时检测这5种致病性弧菌,具有高通量、灵敏性高、重复性好、可控性强、节省样本和时间等优点,对水产品中多种重要致病性弧菌的检测和流行病学调查提供了技术支持。
A rapid and accurate multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus,Vibrio alginolyticus and Vibrio mimicus.Ten specific primers and probes were designed and synthesized based on the species-specific genes and virulence genes of five pathogenic Vibrios.After multiplex PCR amplification,the products were hybridized with microsphere mixtures that have been coupled to gene-specific probes.The suspension array system was used to detect the result of hybridization.The results showed that the detection limit for the five pathogenic Vibrios could reach to 1.8×10^(2) CFU/mL;Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other food borne pathogens tested,which included Escherichia coli,Staphylococcus aureus,Vibrio fluvialis,Salmonella,Listeria and aeruginosa;The method has good repeatability in the detection of five pathogenic Vibrios and the CV was below 8%.The bead-based suspension array method established in this study could rapidly,simultaneously detect five pathogenic Vibrios,and has the advantages of high throughput,high sensitivity,good repeatability,strong controllability,saving samples and time,etc.It provide a technical support for the detection and epidemiological investigation of a variety of important pathogenic Vibrios in aquatic products.
作者
郭容
董晓妹
别闯南
王权
韩先干
叶建强
蒋蔚
GUO Rong;DONG Xiao-mei;BIE Chuang-nan;WANG Quan;HAN Xian-gan;YE Jian-qiang;JIANG Wei(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;Key Laboratory of Jiangsu Preventive Vete rinary Medieine,Yangzhou University,Yangzhou 225009,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第7期805-813,共9页
Chinese Veterinary Science
基金
兵团财政科技计划项目(2020AB025-03)
国家重点研发计划政府间国际科技创新合作重点专项(2018YFE0102200)
上海市科学技术委员会科研计划项目(13DZ05022702)
上海市科技兴农重点攻关项目(2020-02-08-00-08-F01489)
中央级公益性科研院所基本科研业务费专项(2020JB07)。
关键词
弧菌
种特异性基因
毒力基因
液相芯片
多重检测
Vibrio
species-specific gene
virulence gene
Luminex xMAP
multiple detection
