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

【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。

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

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.