肠出血性大肠杆菌变种的RAPD分型研究

食品科学 ›› 2012, Vol. 33 ›› Issue (17): 184-187.

肠出血性大肠杆菌变种的RAPD分型研究

翟平平1，李嘉文1，王芳2，熊燕2，李睿1,*

1.武汉工业学院生物与制药工程学院 2.武汉市疾病预防控制中心

收稿日期:2011-07-25 修回日期:2012-08-02 出版日期:2012-09-15 发布日期:2012-11-09
通讯作者: 李睿 E-mail:liruiwuhan77@yahoo.com.cn
基金资助:
一种新发现的大肠杆菌致病菌志贺毒素调控机制研究

RAPD Typing of Enterohemorrhagic E. coli Mutants

Received:2011-07-25 Revised:2012-08-02 Online:2012-09-15 Published:2012-11-09
Contact: Rui Li E-mail:liruiwuhan77@yahoo.com.cn

摘要/Abstract

摘要： 对4株肠出血性大肠杆菌(EHEC)进行随机扩增多态性(RAPD)分析，并结合主要毒力基因如eae和hly，以及志贺毒素滴度，探讨RAPD实验对大肠杆菌致病菌进行基因分型结果的可靠性。实验菌株中有两株变种携带stx基因但不能正常表达志贺毒素，其中一株变种EC169与另两株正常表达志贺毒素的O157菌株具有相似的扩增图谱，而另一株非O157变种EC130与其他菌株聚类明显不同。从20条随机引物中筛选出了重复性强且具有多态性的随机引物G2、G7、G8、G11、G12，可用于大肠杆菌致病菌快速鉴别和食物中毒溯源。

关键词: 肠出血性大肠杆菌(EHEC), 志贺毒素, 随机扩增多态性(RAPD), 基因分型

Abstract: Four enterohemorrhagic Escherichia coli (EHEC) strains were analyzed by random amplified polymorphic DNA (RAPD) method, and the reliability of genotyping of pathogenic E. coli by RAPD was discussed in terms of the major toxic genes, eae and hly and Shiga toxin titer. Two of the test strains carried stx genes but did not express Shiga toxins. Of them, one mutant strain, O157 EC169, had a similar RAPD profile with the other two O157 strains which could express Shiga toxins, while the other mutant, O157 EC130 revealed significant cluster difference from other strains. Several primers with strong repeatability and polymorphism that are available for fast identification of E. coli pathogenic bacteria and the food poisoning traceability were selected from 20 random primers.

Key words: enterohemorrhagic Escherichia coli (EHEC) , Shiga toxins, random amplified polymorphic DNA (RAPD) , genotyping

中图分类号:

R155.5

翟平平1，李嘉文1，王芳2，熊燕2，李睿1,*. 肠出血性大肠杆菌变种的RAPD分型研究[J]. 食品科学, 2012, 33(17): 184-187.

参考文献

[1] MAINIL J G, DAUBE G. Verotoxigenic Escherichia coli from animals, humans and foods: who’s who?[J]. J Appl Microbiol,2005, 98(6): 1332-1344.
[2] PERNA, N., PLUNKETT, G., III, BURLAND, V. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature,2001,409:529-533.
[3] WILLIAMS J,KUBELIK A,LIVAK K,et a1. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J]. Nucleic Acids Res,1990,18(22):6531-6535.
[4] WELSH J,MCCLELLAND M. Fingerprinting genomes using PCR with arbitrary primers[J]. Nucleic Acids Res,1990,18(24):7213-
7218.
[5] MOORE J E, WATABE M, MILLAR B C, et al. Molecular characterization of verocytoxigenic Escherichia coli O157: H7 by random amplification of polymorphic DNA (RAPD) typing [J]. Br J Biomed Sci, 2008, 65(3):161-163.
[6] 张颖华,王小光,丁克颖,等. RAPD在大肠埃希菌O157同源性分析中的应用[J]. 中国卫生检验杂志,2003,20(3):470-471.
[7] 薛峰,徐飞,张小荣,等. 空肠弯曲菌PCR-RAPD分子亚分型及其毒力相关基因分析[J]. 农业生物技术学报,2010,3:586-591.
[8] 戴诗皎,李睿,刘志国,等. 产志贺毒素突变株的毒力分析[J]. 中国酿造,2010,10:145-147.
[9] 汪铭书,程安春,李淑梅,等. RAPD用于鸭疫里默氏杆菌鸭源致病性大肠杆菌和鸭沙门氏菌的遗传相似性及聚类分析的初步研究[J]. 中国兽医杂志,2007,43(9):9-11.
[10] 李亚玲,韩国民,何沙娥,等. 基于DNA分子标记数据构建系统进化树的新策略[J]. 生物信息学,2008,4:168-170.
[11] LIN C K, LIN J C. Development of PCR premiers based on a fragment from randomly amplified polymorphic DNA for the detection of Escherichia coli O157: H7 /NM [J]. Molecullar Cellular Probes, 2007, 21(3):182 -1891.
[12] LAING C, PEGG C, YAWNEY D, et al. Rapid determination of Escherichia coli O157: H7 lineage types and molecular subtypes by using comparative genomic fingerprinting[J]. Appl. Envir. Microbio, 2008, 74 (21) :6606-66151.
[13] 薛峰,徐飞,张小荣,等. 空肠弯曲菌PCR-RAPD分子亚分型及其毒力相关基因分析[J]. 农业生物技术学报,2010,18(3):586-591.
[14] 蒋国芳,陆敢,黄琨,等. 用RAPD 标记研究蚱属五个种间的亲缘关系[J]. 昆虫学报, 2002, 45 (4):499-502.
[15] 姜自锋,林乃铨,徐梅. RAPD技术及其应用中的一些问题[J]. 福建农林大学学报,2002,31(3):356-360.