环介导等温扩增联合横向流动试纸条可视化检测海豚链球菌方法的建立  

Visual Detection of Streptococcus iniae Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

作　　者：王瑞娜[1] 周前进[1] 陈炯[1] 

机构地区：[1]宁波大学生物与海洋科学系,宁波315211

出　　处：《农业生物技术学报》2014年第12期1584-1594,共11页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2012AA092001;No.2012AA020101);宁波大学学科项目(No.xk11341)

摘　　要：海豚链球菌(Streptococcus iniae)是一种革兰氏阳性球菌,呈β溶血,可感染多种淡水和海洋鱼类。本研究利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)进行核酸扩增,通过横向流动试纸条方法 (lateral flow dipstick,LFD)实现检测,建立了一种可应用于海豚链球菌快速检测的LAMP-LFD技术。该技术以海豚链球菌促旋酶B亚单位(gyrase subunit B,gyr B)基因为检测靶标,设计3对引物进行由生物素标记的LAMP扩增反应,产物经异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的探针杂交后,在LFD上完成检测。经优化的核酸扩增最适条件为65℃反应30 min,在此条件下,阳性扩增起始时间与模板浓度之间呈典型的线性相关性。从核酸扩增反应开始到LFD显色,整个检测时程只需40 min左右,比常规PCR技术缩短约2 h。LAMP-LFD能特异性地检出海豚链球菌,针对病原纯培养物的检测灵敏度为8.70×101cfu/m L,是LAMP检测的10倍、常规PCR检测的100倍。以灵敏度浓度(8.70×101cfu/m L)的海豚链球菌基因组DNA为模板进行的LAMP-LFD结果显示该方法具有良好的重复性。针对人工污染花鲈(Lateolabrax japonicus)肝组织的检测灵敏度为4.35×103cfu/m L,同样为常规PCR检测方法的100倍。利用本方法可成功从患病花鲈的组织样品中检测出海豚链球菌,检测结果与常规的细菌分离鉴定方法结果一致。因此,利用LAMP-LFD能特异、准确、高效地检测出海豚链球菌,而且操作简单、费用低、耗时短,有望成为海豚链球菌的常规检测方法。Streptococcus iniae is a species belonging to Gram-positive coccus and produces beta hemolysis, which can infect a broad range of freshwater and marine fish species and lead to seriously economic losses in the aquaculture industry worldwide. Thus, a diagnostic method for rapid and accurate detection of S. iniae was urgently needed to effectively control the spread of streptococcicosis caused by S. iniae. In the present study, a LAMP-LFD method was developed to rapid detection of S. iniae, based on the loop-mediated isothermal amplification （LAMP） and visualization by a lateral flow dipstick （LFD）. Three pairs of primers, i.e., gyrB-F3, gyrB-B3, gyrB-FIP, gyrB-BIP, gyrB-LF and gyrB-LB, were designed to recognize the gyrase subunit B （gyrB） gene of S. iniae;and among which the forward inner primer （gyrB-FIP） was biotinylated. A DNA probe（gyrB-HP） labeled by fluorescein isothiocyanate （FITC） was also designed specifically to recognize the gyrB gene. The biotinylated LAMP was performed and the LAMP product was hybridized with FITC-labeled probe and detected by LFD. The optimized assay could detect S. iniae by incubation at 65＆#176; C for 30 min, and under such conditions, the initiation time for positive amplification gradually increased as the concentration of template decreased, showing a typical linear relationship. Starting from isothermal amplification, the whole detection procedure needed only 40 min, which reduced by 2 h compared to conventional PCR assay. The LAMP-LFD could specifically detect S. iniae isolate and no amplification was observed from other pathogenic bacteria, e.g., the Streptococcus （i.e., Streptococcus dysgalactiae subsp., and et al）, multiple Gram-positive bacteria （i.e., Staphylococcus aureus, Nocardia seriolae, and et al）, and multiple Gram-negative bacteria （i.e., Vibrio harveyi, Pseudomonas aeruginosa, and et al）. The detection limit of LAMP-LFD for pure culture of S. iniae was 8.70×10^1 cfu/mL （1.74 cfu/reaction）, which was 10 fold higher than

关 键 词：海豚链球菌 环介导等温扩增技术(LAMP) 横向流动试纸条(LFD) 特异性 灵敏度 

分 类 号：S511.03[农业科学—作物学]