Contamination of foods and sources of the emerging diarrheagenic Fec ericbia mallet foreshadowspandemics

食品污染和新出现的致腹泻性粪球菌来源预示着流行病的发生

• 批准号: 18602001
• 负责人: NISHIKAWA Yoshikazu
• 金额: $ 2.55万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18602001/
• 关键词: darrheagenic; Escherichia coli; real-time PCR; multiplex; food; screening; epidemiology; contamination

Diarrheagenic Escherichia coli (DEC) comprise a variety of pathotypes: enteropathogenic E. coli (EPEC); shigatoxin-producing E. coil (STEC); enterotoxigenic E. coli (ETEC); enteroinvasive E. coli (EIEC); enteroaggregative E. call (EAggEC); and diffusely adhering E. coil (DAEC). However, the source and routes of infection have not been clarified because these organisms are scarcely detected among numerous coliform bacteria. To exhaustively detect DEC in food, we have developed multiplex real-time PCR assays against enterovirulent genes (eae, stx1, stx2, elt, est, virB, aggR, astA and afaB). Primers and TaqMan probes were designed to amplify and quantify the genes in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 96 DEC strains, 2 strains of Vibrio cholerae El Tor Ogawa, and Shigella dysenteriae serotype 1. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, ground meat samples and enrichment broths were spiked with DEC. Detection limits varied from 3.6×10^2 to 6.0×10^3 CFU/ml, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU/10g) were found to be positive by screening the enrichment broth using multiplex real-time PCR. As a preliminary test, 148 food samples were exhaustively assayed by multiplex real-time PCR; 41 samples (28%) were positive for DEC pathotypes. The present multiplex real-time PCR system allows the efficient and simultaneous determination of various DEC pathotypes in food.

致泻性大肠杆菌（DEC）包括多种致病型：肠致病性E.大肠杆菌（EPEC）、产志贺毒素E. coil（STEC）;肠毒素E.大肠埃希菌（ETEC）、肠侵袭性E.大肠杆菌（EIEC）、肠聚集性E.呼（EAggEC）;和广泛粘附的E。线圈（DAEC）。然而，感染的来源和途径尚未明确，因为在众多的大肠菌群中几乎没有检测到这些微生物。为了彻底检测食品中的DEC，我们已经开发了针对肠毒力基因（eae，stx 1，stx 2，elt，est，virB，aggR，astA和afaB）的多重实时PCR检测。设计引物和TaqMan探针以在相同PCR循环条件下在双链或三链反应中扩增和定量基因。使用96株DEC菌株、2株霍乱弧菌El Tor Ogawa菌株和志贺菌血清型1确认了专属性。荧光阈值循环和DNA浓度相关的决定系数大于0.99。随后，将DEC掺入碎肉样品和富集肉汤中，检测限从3.6×10^2到6.0×10^3 CFU/ml不等，具体取决于靶基因。通过多重实时PCR筛选增菌肉汤，发现所有添加各种DEC（超过10 CFU/10 g）的肉类样品均为阳性。作为初步测试，148个食品样品进行了详尽的多重实时PCR检测，41个样品（28%）是阳性的DEC致病型。本多重实时PCR系统可以有效地和同时确定各种DEC致病型的食品。

项目成果

Prevalence of enteric bacteria that inhibit growth of enterohaemorrhagic Escherichia coli 0157 in humans

抑制人类肠出血性大肠杆菌 0157 生长的肠道细菌的流行情况

• 发表时间: 2007
• 期刊: Epidemiology and Infection 135・1
• 作者: Toshima;H.;Y.;Hachio;M.;Ikemoto;Y.;Ogasawara;J.;Hase;A.;Takahashi;K.;Masaki;H.;Nishikawa;Y.
• 通讯作者: Y.

Epithelial cells secrete interleukin-8 in response to adhesion and invasion of diffusely adhering Escherichia coli lacking Afa/Dr genes

• DOI: 10.1111/j.1348-0421.2006.tb03781.x
• 发表时间: 2006-01-01
• 期刊: MICROBIOLOGY AND IMMUNOLOGY
• 影响因子: 2.6
• 作者: Meraz, IM;Arikawa, K;Nishikawa, Y
• 通讯作者: Nishikawa, Y

リアルタイムPCRによる食品中の下痢原性大腸菌(EHEC, EPEC, ETEC)同時検出法

使用实时 PCR 同时检测食品中的致泻性大肠杆菌 (EHEC、EPEC、ETEC)

• 发表时间: 2006
• 作者: 日高 あゆみ;法橋 朋子;小笠原 準;長谷 篤;西川 禎一
• 通讯作者: 西川 禎一

下痢原性大腸菌検出方法

致泻性大肠杆菌检测方法

• 发表时间: 2007

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escheriehia coli from calves.

来自小牛的非典型肠病性大肠杆菌和非 O157 产志贺毒素大肠杆菌的 eae 和 stx 基因变体。

• 发表时间: 2007
• 期刊: Lett. Appl. Microbiol. 45
• 作者: Wani;S. A.;Hussain;I.;Nabi;A.;Fayaz;I.;and Nishikawa;Y.
• 通讯作者: Y.