环介导等温扩增联合横向流动试纸条可视化检测志贺氏菌  

Visual detection of Shigella based on loop-mediated isothermal amplification combined with a lateral flow dipstick

作　　者：李尚阳 周前进[1] 张严峻[2] 潘军航[2] 陈炯[1] 

机构地区：[1]宁波大学生物与海洋科学系,浙江宁波315211 [2]浙江省疾病预防控制中心,浙江杭州310051

出　　处：《微生物学通报》2016年第7期1616-1626,共11页Microbiology China

基　　金：国家星火计划(No.2014GA701007);浙江省重大科技专项重点社会发展项目(No.2013C03045-1);宁波市科技创新团队项目(No.2015C110018);宁波市科技富民项目(No.2016C10042);宁波大学学科项目(No.XKL14D2083)~~

摘　　要：【目的】将环介导等温扩增技术（LAMP）与横向流动试纸条（LFD）联合应用,建立一种可应用于志贺氏菌快速检测的LAMP-LFD技术。【方法】以福氏志贺氏菌的侵袭性质粒抗原H（ipa H）基因为检测靶标设计3对特异性引物（其中上游内引物Sfl-ipa H-FIP由生物素标记）,进行LAMP反应;同时设计1条异硫氰酸荧光素（FITC）标记的探针Sfl-ipa H-HP,与获得的LAMP产物进行特异性杂交,杂交产物经LFD完成检测。【结果】优化后的LAMP反应条件为63℃ 40 min,加上LFD结果判读共需50 min。LAMP-LFD方法能够特异性检测出福氏志贺氏菌,而对肠炎沙门氏菌等其它4种导致腹泻的致病菌和创伤弧菌等5种常见食物源性致病菌,以及4株不同大肠杆菌的检测结果呈阴性。该方法针对福氏志贺氏菌的检测灵敏度为1.0×10^2 CFU/m L或4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×10^2 CFU/m L,是以LAMP外引物Sfl-ipa H-F3/Sfl-ipa H-B3的常规PCR方法的100倍。【结论】建立的LAMP-LFD技术具有操作简单、检测快速准确、检测成本低等优点,有望在志贺氏菌的常规监测和即时检测中被普及使用。[Objective] To develop an LAMP-LFD method to detect rapidly Shigella spp., based on nucleotide enrichment by a loop-mediated isothermal amplification（LAMP） and chromatographic visualization by a lateral flow dipstick assay. [Methods] Three pairs of primers were designed based on conserved regions of the invasive plasmid antigen H（ipa H） gene of Shigella flexneri and used in LAMP reaction, among which the forward inner primer Sfl-ipa H-FIP was biotinylated. Similarly, a fluorescein isothiocyanate（FITC）-labeled probe Sfl-ipa H-HP was designed to specifically hybridize with LAMP products. And the hybridized products were visually detected by LFD. [Results] The LAMP-LFD method to detect Shigella spp. was developed. The optimized condition for LAMP reaction was at 63 ℃ for 40 min; and plus visually detecting by LFD, it was approximately 50 min. The LAMP-LFD method discriminated S. flexneri from another 4 pathogenic bacteria causing diarrhea（Salmonella enteric, Campylobacter jejuni, Yersinia enterocolitica, and Vibrio cholera） and 5 common food-borne pathogens（V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus, and V. alginolyticus）, and 4 different Escherichia coli strains. The detection limit of LAMP-LFD was 1.0×102 CFU/m L for Shigella pure cultures（equivalent to 4 CFU per reaction）, which was 100 times lower than that of the conventional PCR method using primers Sfl-ipa H-F3/Sfl-ipa H-B3. In the case of artificially contaminated Common carp intestinal tissue, the detection limit was 5×102 CFU/m L（equivalent to 20 CFU per reaction）. [Conclusion] This rapid and accurate LAMP-LFD method is a promising alternative in the routine surveillance and point-of-care test of Shigella spp.

关 键 词：志贺氏菌 侵袭性质粒抗原H基因 环介导等温扩增技术 横向流动试纸条 检测 

分 类 号：R378[医药卫生—病原生物学]