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

发布时间：2018-06-13 01:13

本文选题：志贺氏菌 + 侵袭性质粒抗原H基因　；参考：《微生物学通报》2016年07期

【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°C 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。
[Abstract]:[objective] to combine the ring mediated isothermal amplification (Lamp) technique with the lateral flow test strip (LFDs), A LAMP-LFD technique for rapid detection of Shigella flexneri was established. [methods] three pairs of specific primers (including upstream primer Sfl-ipa H-FIP) were designed based on Hapipa H, an invasive plasmid antigen of Shigella flexneri. Labeled with substance, lamp reaction was carried out. At the same time, a probe Sfl-ipa H-HPlabeled with fluorescein isothiocyanate was designed and hybridized specifically with the obtained lamp product. [results] the optimized conditions of lamp reaction were 63 掳C 40 min and 50 min. LAMP-LFD was used to detect Shigella flexneri specifically. The results were negative for 5 common foodborne pathogens such as Salmonella enteritidis and Vibrio vulnificus, and 4 strains of Escherichia coli. The sensitivity of this method for detection of Shigella flexneri was 1.0 脳 10 ~ 2 CFU / mL or 4 CFU / reaction. The sensitivity of detection for artificially contaminated carp intestinal tissue is 5.0 脳 10 ~ 2 CFU / mL, which is 100 times higher than that of conventional PCR method using lamp outer primer Sfl-ipa H-F3 / Sfl-ipa H-B3. [conclusion] the established LAMP-LFD technique has the advantages of simple operation, rapid and accurate detection, low detection cost, and so on. It is expected to be widely used in routine surveillance and immediate detection of Shigella.
【作者单位】：宁波大学生物与海洋科学系;浙江省疾病预防控制中心;
【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)~~
【分类号】：R378

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