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微生物学通报

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
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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)


Visual detection of Shigella based on loop-mediated isothermal amplification combined with a lateral flow dipstick
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    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 (ipaH)基因为检测靶标设计 3对特异性引物(其中上游内引物Sfl-ipaH-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-ipaH-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 1.0×102 CFU/mL或 4 CFU/反应,针对人工污染鲤鱼肠组织的检测灵敏度是5.0×102 CFU/mL,是以LAMP外引物 Sfl-ipaH-F3/Sfl-ipaH-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 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 (ipaH) gene of Shigella flexneri and used in LAMP reaction, among which the forward inner primer Sfl-ipaH-FIP was biotinylated. Similarly, a fluorescein isothiocyanate (FITC)-labeled probe Sfl-ipaH-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 °C 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/mL 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-ipaH-F3/Sfl-ipaH-B3. In the case of artificially contaminated Common carp intestinal tissue, the detection limit was 5×102 CFU/mL (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.

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李尚阳,周前进,张严峻,潘军航,陈炯. 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[J]. 微生物学通报, 2016, 43(7): 1616-1626

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  • 在线发布日期: 2016-07-01
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