弗氏柠檬酸杆菌RPA-LFD快速检测方法的建立及应用
Establishment and application of a method for rapid detection of Citrobacter freundii by recombinase polymerase amplification(RPA)combined with lateral flow strips(LFD)
王姝然 1范厚勇 2徐嘉楠 2周天琦 1许丹 1郑跃平2
作者信息
- 1. 上海海洋大学水产与生命学院,上海 201306;上海海洋大学国家水生动物病原库,上海 201306;上海海洋大学农业农村部淡水水产种质资源重点实验室,上海 201306
- 2. 上海市水生野生动植物保护研究中心,上海 200092
- 折叠
摘要
为建立一种针对弗氏柠檬酸杆菌(Citrobacter freundii)敏感性高、特异性强的临床快速检测方法,根据该菌定居因子基因cfa的保守序列,设计特异性引物,采用重组酶聚合酶扩增技术(recombinase poly-merase amplification,RPA),并结合琼脂糖凝胶电泳(AGE)和侧流层析试纸条(LFD)方法进行反应条件的优化及特异性与灵敏度的检测.结果表明:本研究中所建立的弗氏柠檬酸杆菌重组酶聚合酶扩增结合侧向流动试纸条(RPA-LFD)快速检测方法,在38℃条件下扩增20 min后,能特异性地检测到弗氏柠檬酸杆菌,对弗氏柠檬酸杆菌纯培养物和基因组DNA的最小检出限分别为1.5×102 CFU/mL和200 fg/μL,是常规PCR(cfa引物)检测灵敏度(20pg/μL)的100倍;利用所建立的RPA-LFD法与常规PCR同时检测杂交鲟攻毒试验样本,两种方法的检测结果一致.研究表明,本研究中建立的弗氏柠檬酸杆菌RPA-LFD方法,操作简便、反应迅速、特异性好、灵敏度高,并且不需要昂贵的仪器,该方法可为未来弗氏柠檬酸杆菌细菌性疾病的早期诊断提供更有效的技术支持.
Abstract
In order to establish a rapid detection method for Citrobacter freundii based on recombinase polymerase amplification(RPA)technology,the specific RPA primers were designed based on the conserved sequence of the cffa gene of C.freundii,the reaction conditions were optimized,and the specificity and sensitivity were evaluated by recombinase polymerase amplification(RPA)combined with agarose gel electrophoresis(AGE)and lateral flow chromatography(LFD).The results showed that C.freundii was detected within 20 min of amplification at 38 ℃ by the established RPA-LFD rapid detection method,with the minimum limit of detection(LOD)of the C.freundii pure culture and genomic DNA of 1.5×102 CFU/mL and 200 fg/μL by RPA-LFD method,100 times higher sensi-tivity than that by conventional PCR(cfa primer).The consistent results were observed when sturgeon hybrid in-fected with C.freundii clinical samples was simultaneously detected using both the established RPA-LFD and con-ventional PCR methods.The research indicated that the RPA-LFD method for C.freundii established in this study is simple,rapid,specific and sensitive,and does not require expensive instrumentation,which can provide more effective technical support for the early diagnosis of bacterial diseases caused by C.freundii in the future.
关键词
弗氏柠檬酸杆菌/快速检测/重组酶聚合酶扩增技术/侧向流动试纸条Key words
Citrobacter freundii/rapid detection/recombinase polymerase amplification technique/lateral flow test strips引用本文复制引用
基金项目
上海市水生野生动植物保护研究中心委托项目(沪水野研专项2022-01号)
出版年
2024
NETL
NSTL
维普
万方数据