大肠杆菌O157的志贺毒素基因克隆和测序

将一株大肠杆菌O157PCR扩增stx2基因全长并克隆测序。该菌株stx2基因与GenBank数据库收录的stx2基因最高同源性为98%,在3个核苷酸位点存在基因突变。采用邻位相连法构建进化树,序列分析结果表明O157为stx2C基因亚型。了解大肠杆菌O157的基因突变情况,并为开发大肠杆菌分子检测方法提供了参考。     

产志贺毒素大肠杆菌O26多重双启动寡核苷酸引物-PCR检测方法的建立

为建立一种三重DPO-PCR方法用于食品样品中的产志贺毒素大肠杆菌O26的检测。以志贺毒素stx1和stx2、O抗原基因wzx O26特异性和实际检测效果,设计引物,构建三重DPO-PCR反应体系,进行特异性、灵敏度、模拟样品验证和实际样品验证。结果表明,三对DPO引物对退火温度不敏感,在49~69℃之间均能发生扩增,且引物之间干扰较小,具有较高的特异性,除目的基因外非目标细菌均无扩增条带出现,纯菌灵敏度检测表明,三重DPO-PCR方法对O26的最低检测限为3.8×10^3 cfu/g。在模拟样品和实际样品中具有良好的检测效果。本研究基于DPO引物构建的三重DPO-PCR方法具有效率高,特异性强,不受退火温度限制等优点,可用于食品样品中产志贺毒素大肠杆菌O26的快速准确检测提供一种高效的辅助检测方法。     

食品中非O157大肠杆菌志贺毒素基因序列分析

对一株从食品中分离的非O157产志贺毒素1型大肠杆菌(EC6)进行研究。将该菌株所产志贺毒素Stx1用 PCR扩增stx1 基因全长并克隆测序,其stx1 基因与GenBank 数据库收录的stx1 基因最高同源性为99%,表明EC6 发生了一定程度的基因突变。采用邻位相连法构建进化树,结果表明EC6 为stx1 基因亚型。     

伊犁地区肉牛源产志贺毒素大肠杆菌的血清型和ERIC-PCR基因型研究

目的:了解产志贺毒素大肠杆菌在伊犁地区肉牛养殖环境和加工环节中的污染状况及其遗传多样性,为产业链中食源性致病性大肠杆菌的风险监测和控制提供基础数据。方法:采用传统方法和PCR方法对养殖环节的饲草料和粪便及屠宰环节的553份样品进行产志贺毒素大肠杆菌的污染调查,对分离鉴定的产志贺毒素大肠杆菌进行7种常见血清型（O145、O157、O45、O103、O111、O26、O121）的PCR检测和ERIC-PCR的基因分型。结果:检测553份样品中有39株编码志贺毒素基因,产志贺毒素大肠杆菌（STEC）的检测率是7.1%。常见血清型PCR检测中血清型O111有2株菌,检出率是5.1%;O145有5株菌,检出率是12.8%。ERIC-PCR基因分型产志贺毒素大肠杆菌有10种基因亚型,分成3簇,A簇有23株菌,相似性在59%¹00%,表明这些菌株之间的亲缘关系较近。结论:伊犁地区肉牛粪便是产志贺毒素大肠杆菌的污染源,这些菌株的亲缘关系较近。      

多重PCR法检测产志贺毒素性大肠杆菌的 4个血清型

目的建立多重PCR法检测产志贺毒素性大肠杆菌(shiga toxin-producing Escherichia coli, STEC)主要血清型O26、O145、O45和O121的分析方法。方法根据GenBank登录序列,设计扩增O抗原翻转酶(wzx)基因的引物,建立PCR方法,并以STECO26、O145、O45和O121基因组为模板,检验多重PCR的灵敏度和特异性。使用建立的PCR,检测牛胴体表面拭子,阳性扩增条带送测序,以验证PCR扩增的可靠性。同时将阳性扩增样品,涂布显色平板,分离靶标血清型细菌。结果本研究成功建立STEC O26、O145、O45和O121的多重PCR方法, PCR循环参数中退火温度为60℃,扩增片段分别为249、353、890和587 bp。多重PCR直接检测O26、O145、O45和O121时,最低检测限介于10~3～10~4 CFU/mL,而增菌后再检测,最低检测限均为1CFU/m L。多重PCR用于其他血清型STEC,和非大肠杆菌扩增时,均未扩增出目的条带,只有O26、O145、O45和O121能够扩增出相应条带。当使用多重PCR直接检测胴体擦拭子时,阳性率为5.45%(3/55),主要为O26、O145血清型;增菌后检测阳性率为7.27%(4/55),主要为O26、O145和O121血清型。阳性PCR扩增样品,成功分离到O26两株、O145和O121各一株。分离菌株具有典型大肠杆菌的生化特性,携带STEC代表性毒力因子志贺毒素和紧密素,且具有多重耐药性。结论以STECO26、O145、O45和O121的wzx基因为检测靶标,成功建立多重PCR方法,灵敏度和特异性良好,与细菌分离联合使用,可减少工作量,精准分离目的病原菌。     

利用EHEC琼脂平板从食物中分离检测产志贺毒素大肠杆菌

为利用肠出血性大肠杆菌(EnterohaemorrhagicEscherichiacoli,EHEC)琼脂平板,从食物中分离、检测产志贺毒素大肠杆菌(shigatoxin-producingEscherichiacoli,STEC),采用STEC毒力基因(hlyA基因)检测溶血素的产生,建立了一种利用EHEC琼脂平板快速、准确地从食物中分离、检测STEC的方法。该方法可检测STEC不同血清O157∶H7、O26、O111。     

检测产志贺毒素大肠杆菌的菌落PCR方法

针对产志贺毒素大肠杆菌的毒力基因stx 设计特异性引物,并建立一种菌落PCR 方法。菌落PCR 模拟实验证实,该方法特异性强,能良好的扩增出O157 的stx1 和stx2 基因,而普通大肠杆菌、蜡样芽孢杆菌、金黄色葡萄球菌则无PCR 扩增产物。应用分子检测初筛、选择性培养、菌落PCR 相结合的方法,检测实际食品样品,分离检测到一株携带 stx1 的产志贺毒素大肠杆菌。本实验建立的菌落PCR 方法可应用于食品检验。     

关于产志贺毒素大肠杆菌O26的解读

正近日,据美国食品安全新闻网消息,由美国墨西哥风味连锁餐厅Chipotle食物中毒引发的产志贺毒素大肠杆菌O26疫情在美国蔓延,导致20人住院。美国疾病预防和控制中心(CDC)称,近两年来由产志贺毒素大肠杆菌O26引发的食物中毒事件明显增多,在未来可能引发更多的疫情,尤其是引发溶血性尿毒综合症的病例数量可能会远超过产志贺毒素大肠杆菌O157。专家解读一、产志贺毒素大肠杆菌是全球最重要的新发高致病性食源性病原菌产志贺毒素大肠杆菌(Shiga toxinproducing Escherichia coli,简称STEC)     

大肠杆菌O157志贺毒素1胶乳凝集试剂的研制

根据大肠杆菌933W志贺毒素1(STX1)基因的序列,设计合成寡核苷酸引物,以国内分离的大肠杆菌O157菌株94H的DNA为摸板,经PCR扩增志贺毒素1A亚基基因,扩增的基因克隆到原核表达载体pET-28a,在E.coliBL21中进行表达。用表达的STX1A产物免疫兔子,制备STX1A抗血清,从中提取IgG。合成胶乳,用提取的IgG与胶乳交连反应,研制出了敏感和简便的检测大肠杆菌O157STX1产生的胶乳检测试剂。     

武汉市部分菜场肉类食品中产志贺毒素大肠杆菌污染状况分析

对武汉市部分菜场肉类食品中产志贺毒素大肠杆菌(STEC)污染状况进行分析。方法 2011年7月至2012年10月期间,从武汉市汉口30个菜场和超市共采集样品196份,其中猪肉102份,牛肉60份,禽类34份。通过选择性增菌,提取DNA,PCR扩增stx1、stx2、rfbO157、wzyO157基因,分析食品中产志贺毒素大肠杆菌的污染状况。结果 猪肉、牛肉、禽类食品中非O157型STEC的阳性检出率分别为18.6%、48.4%和2.9%;O157型STEC的阳性检出率分别为13.6%(猪肉)、6.7%(牛肉)和2.9%(禽类)。菜场样品STEC检出率(35.8%)略高于超市样品(32.4%)。结论 武汉市肉类食品中STEC检出率较高,应定期跟踪监测,了解菌株流行和毒力变化状况。     

Comparison of Decontamination Efficacy of Antimicrobial Treatments for Beef Trimmings against Escherichia coli O157:H7 and 6 Non-O157 Shiga Toxin-Producing E. coli Serogroups

Abstract: The decontamination efficacy of 6 chemical treatments for beef trimmings were evaluated against Escherichia coli O157:H7 and 6 non‐O157 Shiga toxin‐producing E. coli (nSTEC) serogroups. Rifampicin‐resistant 4‐strain mixtures of E. coli O157:H7 and nSTEC serogroups O26, O45, O103, O111, O121, and O145 were separately inoculated (3 to 4 log CFU/cm²) onto trimmings (10 × 5 × 1 cm; approximately 100 g) fabricated from beef chuck rolls, and were immersed for 30 s in solutions of acidified sodium chlorite (0.1%, pH 2.5), peroxyacetic acid (0.02%, pH 3.8), sodium metasilicate (4%, pH 12.5), Bromitize^® Plus (0.0225% active bromine, pH 6.6), or AFTEC 3000 (pH 1.2), or for 5 s in SYNTRx 3300 (pH 1.0). Each antimicrobial was tested independently together with an untreated control. Results showed that all tested decontamination treatments were similarly effective against the 6 nSTEC serogroups as they were against E. coli O157:H7. Irrespective of pathogen inoculum, treatment of beef trimmings with acidified sodium chlorite, peroxyacetic acid, or sodium metasilicate effectively (P < 0.05) reduced initial pathogen counts (3.4 to 3.9 log CFU/cm²) by 0.7 to 1.0, 0.6 to 1.0, and 1.3 to 1.5 log CFU/cm², respectively. Reductions of pathogen counts (3.1 to 3.2 log CFU/cm²) by Bromitize Plus, AFTEC 3000, and SYNTRx 3300 were 0.1 to 0.4 log CFU/cm², depending on treatment. Findings of this study should be useful to regulatory authorities and the meat industry as they consider nSTEC contamination in beef trimmings. Practical Applications: Findings of this study should be useful to: (i) meat processors as they design and conduct studies to validate the efficacy of antimicrobial treatments to control pathogen contamination on fresh beef products; and (ii) regulatory agencies as they consider approaches for better control of the studied pathogens.     

不对称PCR技术结合免疫层析法快速检测产志贺毒素大肠埃希氏菌

产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli,STEC)可分泌志贺毒素,致病性强,建立简便快速的STEC检测方法对控制食源性疾病的发生和蔓延极为重要.使用生物素标记STEC的标志性毒力基因stx1与stx2的下游引物进行不对称聚合酶链式反应,获得生物素化的目标单链...     

Detection of Escherichia coli O157 in bovine meat products in northern Italy

Tests for Escherichia coli and E. coli O157 were carried out on meat samples collected from randomly chosen stores throughout the city of Bologna and suburban areas. The samples consisted of 25 g of loose minced beef, sometimes already shaped into meatballs or hamburgers, some of which were mixed with vegetables. The meat was purchased from retail outlets, open market stalls, and supermarket chains during 25 sampling visits from October 2000 to December 2001. For E. coli detection, Tryptone soya broth (TSB) supplemented with novobiocin and C-EC agar were used. Immunomagnetic separation with SMAC-BCIG-CT agar and chromogenic E. coli O 157 agar, API 20E system and agglutination latex test were used to detect E. coli O157; Vero cell assay and polymerase chain reaction (PCR) were used to assess toxin production and the presence of virulence genes.

E. coli were detected in 45 (30.2%) of the 149 samples examined, mainly in the hamburger samples mixed with vegetables and in the loose minced beef. E. coli O157 was found in one sample of hamburger and two samples of hamburger mixed with vegetables (2%) collected from three different butcher's stores between July and October. All the strains of E. coli O157 and most cases of E. coli were found in meat from small retailers.

The three strains of E. coli O157 were positive for verocytotoxin production. PCR analysis revealed genes coding for vt2 and one strain possessed the gene for eae A. Chromogenic E. coli O157 agar was found to be more selective and differential, allowing easier identification of suspected colonies with mixed flora and producing less false-positive colonies.     

蔬菜中大肠杆菌O157的分离与鉴定

对武汉市售蔬菜(50份)进行大肠杆菌O157的检验,经过新生霉素-EC增菌液增菌、免疫磁珠富集、选择性平板培养和血清学鉴定,从一份生菜中筛出1株O157阳性菌株EC9.23。PCR鉴定该菌毒力基因,stx1、stx2、rfbO157基因均为阳性,hly、eae、fliCH7基因均为阴性。说明从武汉市售蔬菜中可检出携带志贺毒素基因的O157菌株。     

Microbiological Quality of Finfish and Shellfish with Special Reference to Shiga Toxin-Producing Escherichia coli O157

ABSTRACT:  The objective of this study was to determine the microbiological quality of fish and shellfish from Kolkata, India, with special emphasis on E. coli O157. Fresh and ice-preserved Labeo rohita , Catla catla , Cirrhinus mrigala , Oreochromis mossambica , Heteropneustes fossilis , Clarias batrachus , and Penaeus monodon were examined for total heterotrophic bacteria and coliform loads and presence of E. coli and E. coli serotype O157 by culture method. While the total plate count of bacteria was within acceptable or marginally acceptable limits for most samples, fishes were contaminated with coliforms, including E. coli , indicating poor hygiene and sanitary conditions. Although E. coli O157 could not be detected, a few samples were contaminated with non-O157 serotypes of enterohaemolysin- and Shiga toxin-producing E. coli , raising public health concern.     

LAMP实时浊度法快速检测食品中产志贺毒素大肠埃希氏菌

针对产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli,STEC)stx1和stx2基因的特异性序列分别设计4条特异性引物,采用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,利用实时浊度仪建立食品中STEC的LAMP实时浊度法快速检测方法。LAMP反应在实时浊度仪63℃恒温下1 h可完成。对方法的特异性、灵敏度、稳定性进行了评价,并在实际样品检测中进行应用。经优化该方法的检测灵敏度可达150拷贝/反应,4株STEC菌株LAMP扩增结果与其基因型一致,其他21株非STEC菌株均未出现非特异性扩增。395份食品样品检出STEC阳性68份(阳性率为17.2%),所检食品类别中畜产品阳性率最高(达31.3%),禽产品、水产品和可生食蔬菜均有少量阳性样品检出,阳性样品中以stx2基因阳性型为主。结果表明,LAMP实时浊度法具有快速、灵敏、特异性强、操作简便的优势,适用于食品中STEC的快速筛查。     

食品中大肠杆菌O157的PCR检测与wzy基因的测序分析

大肠杆菌O157:H7 和O157:H- 是重要食品致病菌。从食品中分离得到一株O157:H7 菌株EC5。将该菌株PCR 扩增wzy 基因全长并克隆测序。结果表明,wzy 基因序列与大肠杆菌O157:H7 标准株EDL933 有100% 同源性。从NCBI GenBank 数据库下载O157 菌株wzy 基因序列,针对保守序列设计引物并扩增O157 菌株和非O157 菌株。PCR 结果表明,wzy 基因特异性强,可作为检测O157 菌株的标志基因。本研究为开发大肠杆菌O157 的分子检测技术提供新的研究思路。