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 polymerase 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(20 pg/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。
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 cfa 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 sensitivity than that by conventional PCR(cfa primer).The consistent results were observed when sturgeon hybrid infected with C.freundii clinical samples was simultaneously detected using both the established RPA-LFD and conventional 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.
作者
王姝然
范厚勇
徐嘉楠
周天琦
许丹
郑跃平
WANG Shuran;FAN Houyong;XU Jianan;ZHOU Tianqi;XU Dan;ZHENG Yueping(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;National Pathogen Collection Center for Aquatic Animals,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;Shanghai Aquatic Wildlife Conservation and Research Center,Shanghai 200092,China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2024年第2期241-249,共9页
Journal of Dalian Ocean University
基金
上海市水生野生动植物保护研究中心委托项目“中华鲟主要细菌性疾病的预警和防控”(沪水野研专项2022-01号)。
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
Citrobacter freundii
rapid detection
recombinase polymerase amplification technique
lateral flow test strips
