fliC基因在快速分型鉴定伤寒沙门菌和甲型副伤寒沙门菌的应用

目的:探讨沙门菌1相鞭毛蛋白抗原fliC基因在快速分型鉴定伤寒沙门菌和甲型副伤寒沙门菌的应用。方法:根据伤寒沙门菌和甲型副伤寒沙门菌1相鞭毛蛋白fliC-d和fliC-a基因以及菌体抗原rfbS基因的核酸序列,设计针对fliC-d、fliC-a及rfbS基因的3对特异性引物,采用多重PCR法进行检测。结果:实验结果显示,伤寒沙门菌和甲型副伤寒沙门菌分别在750 bp和329 bp处扩增出2条特异性目的条带,在258 bp处扩增出1条相同条带,非伤寒沙门菌株和甲型副伤寒沙门菌株均为阴性。结论:fliC基因检测可用于伤寒沙门菌和甲型副伤寒沙门菌的分型鉴定,而rfbS基因则无助于分型鉴定。     

多重PCR方法检测大肠杆菌O157:H7的初步研究

目的研究一种快速、特异的检测大肠杆菌O157：H7的多重PCR方法。方法以大肠杆菌O157：H7的O157抗原基因（rfbE基因）、鞭毛H7抗原基因（fliC基因）、粘附素基因（eaeA基因）和志贺样毒素Ⅰ、Ⅱ（SLT-Ⅰ、SLT-Ⅱ基因）为扩增对象，设计能导至目的片段大小不同的5对引物，通过优化条件，建立了可用于粪便检测的大肠杆菌5重PCR。结果在同一扩增体系中，检测了5株不同来源的大肠杆菌O157：H7及27株非大肠杆菌O157：H7细菌。结果表明，该方法能从2株大肠杆菌O157：H7中扩增出rfbE、fliC、eaeA、SLT-Ⅰ、SLT-Ⅱ基因，它们的大小分别为582bp、802bp、487bp、368bp和177bp。而另外3株大肠杆菌O157：H7也能扩增出除eaeA、SLT-Ⅱ基因外的其它3个基因片段。表明该方法可用于检测大肠杆菌O157：H7，还可检测细菌是否含有毒素基因。在检测27株非大肠杆菌O157：H7．除O149出现SLT-Ⅱ扩增产物和01：H7出现flic基因扩增产物外，其它非大肠杆菌O157：H7的扩增结果均为阴性，表明该方法有较高的特异性。当粪便样品经增菌培养12h，用该5重PCR体系能检测出最初接种量为5cfu／g粪便的样品。结论本5重PCR方法具有较高的特异性和敏感性，可迅速、有效地将大肠杆菌O157：H7与其它非大肠杆菌O157：H7相区别，同时还能检测O157：H7中的毒力因子。因此，通过进一步研究，本方法有望应用于临床大肠杆菌O157：H7感染的辅助诊断。     

Molecular serotyping of Salmonella: identification of the phase 1 H antigen based on partial sequencing of the fliC gene

The purpose of this study was to develop a simple and non-labour-intensive molecular method to identify the phase 1 H antigens of Salmonella. The variable region of the flagellin gene, fliC, from 96 Salmonella strains representing 51 different phase 1 H antigens was sequenced in one direction. Unique sequences were found for 45 of the 51 different antigens. We were not able to separate either H:z42 from H:d; H:g, q from H:g, m, q; H:l, w from H:Rl, z40 or H:l, (v),z13 from H:l,z,13. Several phase 2 H antigens were found to be encoded by fliC. Polymorphism, at the subspecies level, was observed in fliC of H:b, H:d, H:z10, H:z and especially H:k. By this method we were also able to confirm that one monophasic strain possesses a new antigen, H:z91. This study shows that sequence-based typing of the phase 1 H antigen of Salmonella is a good alternative to serotyping when strains are non-typable by serological methods.     

动物性食品中大肠杆菌O157:H7多重PCR检测方法的建立

目的:大肠杆菌O157:H7是近年来引发食物中毒事件的重要病原菌,对其建立灵敏、特异的多重PCR检测方法,是确保饮食安全,预防和控制大肠杆菌O157:H7传播的重要手段。方法:根据大肠杆菌O157:H7rfbE和fliC抗原基因的保守序列设计了2对引物,建立并优化了特异性检测大肠杆菌O157:H7的多重PCR体系,扩增产物分别为678 bp(rfbE)和560 bp(fliC)。结果:建立的多重PCR方法可以快速特异地检测出猪肉、牛肉等动物性食品中的大肠杆菌O157:H7污染,人工污染猪肉检测限达到2.2×102cfu/ml。结论:选择两对特异引物的多重PCR方法可简便、快速、特异地实现对动物性食品中大肠杆菌O157:H7的检测。     

Subtyping of pathogenic Escherichia coli strains using flagellar (H)-antigens: serotyping versus fliC polymorphisms

Serotyping of O- and H-antigens is regarded as the gold standard in classification of E. coli for taxonomic and epidemiological purposes similar to the Kaufmann-White scheme for Salmonella enterica. Molecular methods to replace or to support the serotyping have been applied recently. Using the molecular polymorphism of the flagella (H-antigen) gene fliC, more than 220 E. coli strains derived from the E. coli reference collection for O- and H-antigens (The International Escherichia and Klebsiella Centre (WHO)) and from clinical origins have been characterised and a reproducible and clear cut classification with very good correlation to serotyping was found. Only some of the H-antigens have revealed multiple fliC classes and vice versa only rarely some of the fliC classes belong to various H-antigen groups. Since also H-antigen-negative and H-antigen non-typeable strains subjected to fliC classification could be typed properly, it is recommended here to use this rapid approach to classify E. coli under routine conditions rather than using classical serotyping. However, scrotyping--in particular using hyperimmune rabbit sera--will remain the gold standard and the task of Reference Centres only, e.g. for defining novel H-antigen types.