[1]徐兰英)),许汴利)#,马宏).志贺菌6种毒力基因的多重PCR检测[J].郑州大学学报(医学版),2009,(06):1218-1221.
 XU Lanying)),XU Bianli),MA Hong).Detection of six kinds of virulence genes in Shigella species by multiplex PCR[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2009,(06):1218-1221.
点击复制

志贺菌6种毒力基因的多重PCR检测()
分享到:

《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2009年06期
页码:
1218-1221
栏目:
论著
出版日期:
2009-11-01

文章信息/Info

Title:
Detection of six kinds of virulence genes in Shigella species by multiplex PCR
作者:
徐兰英1)2)许汴利2)#马宏2)
1)郑州大学公共卫生学院流行病学教研室 郑州 4500012)河南省疾病预防控制中心 郑州 450016
Author(s):
XU Lanying1)2) XU Bianli2) MA Hong2)
1)Department of Epidemiology,College of Public Health, Zhengzhou University, Zhengzhou 450001 2)Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016
关键词:
志贺菌多重PCR志贺菌肠毒素1基因志贺菌肠毒素2基因侵袭性质粒抗原H基因侵袭相关蛋白基因志贺毒素1基因调控基因
Keywords:
Shigellamultiplex PCRset1senipaHialstx1virA
分类号:
R378.25
摘要:
建立志贺菌6种毒力基因[志贺菌肠毒素1基因(set1)、志贺菌肠毒素2基因(sen)、侵袭性质粒抗原H基因(ipaH)、侵袭相关蛋白基因(ial)、志贺毒素1基因(stx1)及调控基因(virA)]的多重PCR鉴定方法,实现多基因的同时检测。方法:选择志贺菌的6种毒力基因作为靶基因,以A群和C群4株标准及B群和D群4株临床分离株为模型,应用降落PCR方法在1个反应管中同时进行扩增,根据扩增片段大小定性判断结果。通过样本倍比稀释检验多重PCR的灵敏性,并在NCBI网站上用BLAST检索各引物的特异性。另选取河南省2001年至2007年115株志贺菌进行多重PCR检测,以验证多重PCR的可行性。结果:多重PCR能同时检测上述6种毒力基因,与单基因PCR分别检测6种毒力基因相比具有相近的灵敏度与特异性。应用多重PCR方法检测115株志贺菌,除stx1外,其余5种毒力基因的阳性率均在85%以上。结论:多重PCR鉴定志贺菌毒力基因具有简便、快速的特点,适用于毒力基因鉴定和流行病学调查研究。
Abstract:
To develop a method of multiplex PCR to detect virulence genes (set1B, sen, ipaH, ial, stx1,and virA) in Shigella spp.Methods:A total of six kinds of virulence genes were selected as target genes and amplified at the same time in the reaction tube with Touchdown PCR.Reference strains of S.dysenteriae 1(CMCC51054,51342),S. boydii 2(CMCC51149,51225) and 4 clinical isolates of Shigellae(1 strain of S. flexneri 2a and var X,2 stains of S. sonnei) were used in the development of multiplex PCR.DNA template was prepared and 5fold serial dilutions were made for sensitivity test. Primers' specificity was retrieved in the BLAST of NCBI web. Then the result was decided based on molecular weight of the product. A total of 115 strains(2001~2007) Shigellae were used.Results: The 6 virulence genes could be detected with multiplex PCR at the same time.Either 6 virulence genes were detected with multiplex PCR at the same time or detected separately with general PCR,and both ways were sensitive and specific.The positive rates for set1B, sen, ipaH, ial, and virA were all more than 85% in the 115 strains Shigellae.Conclusion: It is convenient and fast to determine virulence genes of Shigella with multiplex PCR.The method is applicable to determine virulence genes for epidemiological investigations.

参考文献/References:

[1]Talukder KA,Islam MA,Dutta DK,et al. Phenotypic and genotypic characterization of serologically atypical strains of Shigella flexneri type 4 isolated in Dhaka, Bangladesh[J]. J Clin Microbiol,2002,40(7):2 490
[2]袁东亚,康龙丽,赵健民,等. 西藏珞巴族15个STR位点遗传多态性研究[J].西安交通大学学报:医学版2006,27(4):319
[3]刘祥印,薛百功,刘睿智,等. 男性不育患者Y染色体AZF区微缺失分析[J].吉林大学学报:医学版,2008,34(2):305
[4]熊燕, 江元山, 陈智,等.志贺菌毒力相关基因的PCR检测分析[J]. 中国卫生检验杂志,2007,17(5):805
[5]马宏,王建丽,黄丽莉,等.志贺菌毒力基因的多重PCR方法鉴定[J].中国卫生检验杂志,2006,16(9):1 038
[6]Thong KL,Hoe SL,Puthucheary SD,et al. Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay[J]. BMC Infect Dis,2005,5(1):8
[7]宋春花,郗园林,张梅喜,等. 郑州市2000年夏秋季腹泻病原菌谱调查[J]. 郑州大学学报:医学版,2002,37(6):840
[8]Noriega FR, Liao FM, Formal SB, et al. Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes[J]. J Infect Dis, 1995, 172(5):1 408
[9]Vargas M,Gascon J,Jimenez De Anta MT,et al. Prevalence of Shigella enterotoxins 1 and 2 among shigella strains isolated from patients with traveler's diarrhea[J]. J Clin Microbiol, 1999,37(11):3 608
[10]孙素霞,王红,王勇,等.福氏志贺菌毒力蛋白IpaC的表达与纯化[J].第一军医大学学报,2003,23(3):230
[11]Menard R,Dehio C,Sansonetti PJ.Bacterial entry into epithelial cells:the paradigm of Shigella[J].Trends Microbiol,1996,4(6):8 220
[12]喻华英,王景林,高丰.志贺毒素及其分子生物学研究进展[J]. 生物技术通讯,2004,15(1):86
[13]Bélanger SD,Boissinot M,Ménard C,et al.Rapid detection of shiga toxinproducing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler[J].J Clin Microbiol,2002, 40(4):1 436
[14]Nataro JP, Seriwatana J, Fasano A, et al. Identification and cloning of a novel plasmidencoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains[J]. Infect Immun,1995,63(12):4 721
[15]张俊琪,钱雪琴,李雪萍,等.福氏志贺菌临床分离株毒力基因的研究[J].中国人兽共患病学报,2007,23(12):1 243

相似文献/References:

[1]宋春花),王颖芳),段广才)#,等.应用抑制性消减杂交筛选志贺菌基因转移耐多药相关基因*[J].郑州大学学报(医学版),2013,(01):63.
 SONG Chunhua,WANG Yingfang,DUAN Guangcai,et al.Screening conjugational transferred multidrug resistancerelated genes in Shigella flexneri by suppression subtractive hybridization[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2013,(06):63.

备注/Memo

备注/Memo:
#通讯作者,男,1958年,硕士,研究方向:分子生物学、传染病及寄生虫病流行病学;Email:xubl@hncdc.com.cn
更新日期/Last Update: 2010-05-14