食品中产志贺毒素大肠杆菌的多重PCR检测

针对产志贺毒素大肠杆菌的stx2、wzy、hlyA的保守序列,设计特异性的引物,建立一种新的多重PCR检验方法.结果显示,该方法特异性强,扩增结果与各参考菌株基因型一致,并能良好的区分出O157菌株和非O157型产志贺毒素大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

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。     

武汉肉类食品中大肠杆菌O157∶H7分离株stx亚型和毒力特征分析

从22 家市场销售的117 份肉类食品中分离出4 株大肠杆菌O157∶H7菌株,经PCR检测这4 株菌的stx、hly、 eae毒力基因均为阳性。采用聚合酶链式反应（polymerase chain reaction,PCR）法对这4 株菌的stx亚型进行鉴定。 3 株菌同时携带stx1、stx2基因,且均为stx1a、stx2a亚型。菌株EC5.11仅携带stx2基因,但在所有的stx2分型PCR反 应中都为阴性。用PCR扩增该菌株stx2基因全长并克隆测序,序列分析结果表明EC5.11志贺毒素基因为stx2c亚型。 采用Vero细胞毒性实验检测这4 株菌产志贺毒素的状况,结果显示这些菌株都具有一定的Vero细胞毒性。     

检测食品中大肠杆菌O157的多重荧光PCR方法的建立

针对大肠杆菌O157的志贺毒素编码基因stx1、stx2和O157血清型的标志基因wzy的保守序列,设计特异性的引物和探针,反复优化反应体系和条件,建立了一种新的多重荧光PCR检验方法.结果显示,该方法灵敏度高,stx1、stx2的扩增效率分别为96.4%和94.6%,实际样品检测时stx1、six2的最低检出限分别为4.2×102cfu/mL和4.2×103cfu/mL.该方法特异性强,扩增结果与各参考菌株基因型一致,能良好的区分出O157菌株和非O157犁大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

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。     

环介导等温核酸扩增技术快速检测食物中的大肠杆菌O157

建立了环介导等温核酸扩增技术(LAMP)检测食源大肠杆菌O157,并对该方法的灵敏度和特异性进行了评价。分别针对大肠杆菌O157三个特异基因rfbE,stx1和stx2的8个独立靶区域设计了外引物、内引物和环引物进行LAMP扩增检测,同时将检测结果与PCR方法做比较。研究结果表明,rfbE,stx1和stx2基因的LAMP方法检测限分别为100,100和10 fg DNA/管,灵敏度是PCR方法的10倍以上;将建立的环介导等温扩增法用于417株食物分离的大肠杆菌的检测,发现LAMP检测rfbE,stx1和stx2基因的灵敏度分别为100%,95.3%和96.3%,对3个靶基因的阴性预测率分别为100%,96.7%和97.1%,特异性和阳性预测率均为100%。结果表明,该方法用于大肠杆菌O157的检测具有特异性强、灵敏度高、操作简便的优越性,在食品安全检测方面具有良好的实际应用前景。     

肠出血性大肠杆菌O157∶H7变种Q蛋白对志贺毒素表达的影响

1株肠出血性大肠杆菌O157∶H7变种EC169菌株,携带stx基因但不表达志贺毒素。通过高效热不对称交错聚合酶链式反应（high-efficiency thermal asymmetric interlaced polymerase chain reaction,hiTAIL-PCR）hiTAILPCR扩增得到EC169 stx1及其上游核苷酸片段并克隆测序,结果表明：EC169 q基因与标准株sakai q基因相比存在6个SNP位点。通过PCR扩增O157∶H7高毒株EC150 q基因全长,并构建表达载体pkk223-q分别转化EC169和低毒株EC157。反转录荧光定量PCR实验结果表明,外源q基因在EC169和EC157重组菌中可高效表达,并引起EC157stx转录水平上调,但EC169重组菌stx转录水平不变。反向乳胶凝集实验结果亦证实EC157重组菌志贺毒素表达量提高,而EC169重组菌志贺毒素表达量不变。Q蛋白变异可能并非EC169志贺毒素不表达的主要原因。     

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DNA与聚苯乙烯微球标记的目标基因的上游引物特异性结合形成结合物,结合物上的生物素因与试纸条检测线上链霉亲和素结合而使检测线显色。通过建立的方法可成功快速检测出携带不同毒力基因的STEC,同时利用该方法对23 株菌株进行stx基因的检测,结果显示该方法可准确检测到含有STEC标志性毒力基因stx的菌株,且表现出良好的特异性。     

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。     

Characterization of Shiga toxigenic Escherichia coli isolated from foods

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.     

Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods

In this study we report on the prevalence and distribution of Shiga toxin-producing Escherichia coli (STEC) in food products of animal origin, collected in the Piedmont region of Italy, as determined by a combination of quantitative PCR (qPCR) protocols, applied directly to the samples, and of culture-dependent isolation and subsequent molecular identification and characterization of isolates. The qPCR protocols were developed and optimized in this study and targeted the rpoB gene (as a marker for total E. coli) and the stx?, stx? and eaeA genes (as markers for potentially virulent E.coli). They were then used to test for STEC in 101 food samples, before and after enrichment. A STEC prevalence of 42% (21/50) for dairy products and 70% (36/51) for meat products was obtained. A total of 54 STEC isolates were recovered from dairy and meat samples, resulting in a prevalence of 36% and 27% in dairy and meat products, respectively, by the culture method. A large number of strains carried the stx? gene (39 out of the 54 STEC strains) compared to strains that carried stx? (30 out of 54); only 11 out of 54 strains contained the eaeA gene, while 14 strains contained both stx? and stx?. Eight of the 54 isolates belonged to the O157 serogroup, and none belonged to serogroups O26, O145, O111 or O103. Strains isolated from meat products were diverse, as determined by Enterobacterial repetitive intergenic consensus-PCR (ERIC), while those isolated from dairy products were more similar and grouped together by cluster analysis. The results of the qPCR approach showed a high prevalence of STEC in dairy and meat based products, mainly fermented, indicating a possible safety risk for these types of food commodities.     

Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.     

Shiga toxin-producing Escherichia coli in ground beef and lamb cuts: results of a one-year study

Shiga toxin-producing Escherichia coli (STEC) have been associated with a broad spectrum of diarrhoeal syndromes. Some of these cases have been attributed to foods of bovine origin or other foods cross-contaminated by beef products or cow manure. The purpose of this study was to determine the pattern of STEC distribution in selected red meats over time. Samples of ground beef and lamb cuts were collected over a 52-week period from 31 different outlets and 25 g portions were assayed for STEC. STEC were isolated from 46/285 (16%) ground beef and 111/275 (40%) lamb samples using an stx PCR screen followed by colony hybridisation. All isolates were tested by PCR for additional STEC virulence markers with 95% of ground beef isolates shown to possess stx(2) and 80% of lamb cutlet isolates shown to possess stx(1) and stx(2). The enterohaemolysin gene (ehxA) was detected in 65% and 53% of ground beef and lamb isolates respectively. Putative enterohaemorrhagic E. coli (EHEC), i.e. STEC possessing the E. coli attaching and effacing gene (eae) were not isolated. The STEC isolates comprised 18 and 15 different serotypes from ground beef and lamb respectively. STEC of serotypes O157, O111 and O26 (common enterohaemorrhagic E. coli serotypes) were not isolated. Serotypes O174 and O91 were the most common serotypes isolated from ground beef samples and O128 and O91 the most common from lamb cutlet samples. The presence of STEC in retail red meats highlights the need for a clearer understanding of STEC in food and human illness to interpret the public health significance of these findings.     

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.     

重组酶聚合酶扩增检测产志贺毒素大肠埃希菌的微流控芯片技术

采用重组酶聚合酶扩增技术（recombinase polymerase amplification,RPA）,开发产志贺毒素大肠埃希菌（Shiga toxin-producing Escherichia coli,STEC）的等温检测方法,结合圆盘式微流控芯片快速检测目标微生物。以stx1和stx2基因为靶点,设计和筛选适宜的RPA检测引物和探针序列,验证了19 株STEC菌株（含9 种stx基因亚型）和21 株非STEC菌株,并采用人工污染的牛肉样品对集成化的微流控芯片进行评价。筛选出检测stx1和stx2基因的高特异性引物、探针组合,与美国农业部微生物实验室技术指南中STEC定量聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）方法相比,目标基因检测的包容性和排他性均为100%。荧光RPA微流控芯片法在20 min内可同时进行32 个反应,可检测STEC菌体灵敏度为9.5×103 CFU/mL。根据GB 4789.6—2016《食品微生物学检验 致泻大肠埃希氏菌检验》的规定经增菌培养后在人工污染牛肉样品中可检测1 CFU/25 g的STEC污染,方法的相对正确度和相对检出水平均为100%。本研究开发了一种荧光RPA微流控芯片检测方法,可检测常见stx基因亚型STEC菌株,操作简单、反应速度快,适用于STEC的高通量快速检测。     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.     

Isolation and characterization of Shiga toxin-producing Escherichia coli O157:H7 and non-O157 from beef carcasses at a slaughter plant in Mexico

The contamination of beef carcasses with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC) obtained from a slaughter plant in Guadalajara, Mexico was investigated. A total of 258 beef carcasses were sampled during a 12-month period. All samples were assayed for STEC by selective enrichment in modified tryptone soy broth supplemented with cefixime, cefsulodin and vancomycin, followed by plating on Sorbitol MacConkey Agar supplemented with cefixime and tellurite (CT-SMAC). Simultaneously, all samples were assayed by immunomagnetic separation (IMS) and plated on CT-SMAC and CHROMagar. The presence of the stx1, stx2, eaeA and hly933 genes, recognized as major virulence factors of STEC, was tested for O157:H7 and non-O157 E. coli isolates by multiplex polymerase chain reaction (PCR). STEC was detected in two (0.8%) samples. One of these STEC isolates corresponded to the serotype O157:H7 showing stx2, eaeA and hyl933 genes. The other isolate corresponded to non-O157 STEC and only had the stx1 gene. Thirteen carcasses (5%) were positive for nonmotile E. coli O157 and 7 (2.7%) were positive for E. coli O157:H7. The presence of O157:H7 and non-O157 STEC on beef carcasses in this slaughter plant in Guadalajara, Mexico, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.     

Short communication: Detection of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle and pigs in Lima, Peru

The aim of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in cattle and pigs as a possible STEC reservoir in Lima, Peru. One hundred and fourteen cattle and 112 pigs from 10 and 4 farms, respectively, were studied. Five E. coli colonies per culture were studied by a multiplex real-time PCR to identify Shiga toxin-producing (stx1, stx2, eaeA), enterotoxigenic (lt, st), enteropathogenic (eaeA), enteroinvasive (ipaH), enteroaggregative (aggR), and diffusely adherent E. coli (daaD). Shiga toxin-producing E. coli were isolated from 16 cattle (14%) but none from pigs. stx1 was found in all bovine isolates, 11 of which also carried eaeA genes (69%); only 1 sample had both stx1 and stx2. Thirteen stx-positive strains were classified as Shiga-toxigenic (81%) using an enzymatic immunoassay, 2 STEC strains were from serogroup O157 (13%), and 7 were sorbitol negative (44%). Enteropathogenic E. coli were detected more frequently in cattle (18%, 20/114) than in pigs (5%, 6/112). To our knowledge, this is the first study on the prevalence of STEC in farms animals in Peru using molecular methods. Further studies are needed in a large number of farms to determine the relevance of these findings and its consequences for public health.