应用通用引物PCR法对葡萄球菌B、C1、C2、C3型肠毒素基因的鉴定与分型研究

本实验采用通用引物P1、P2聚合酶链反应(PCR)法对产肠毒素葡萄球菌的entB、entC1、entC2、entC3基因进行了扩增,结果表明,含有这四种肠毒素基因的葡萄球菌都可以产生595bp的特异性扩增片段,其余菌扩增均为阴性;扩增产物分别用在产物内部只有单一识别位点的限制酶HinfⅠ、BclⅠ、MboⅡ和MboⅡ酶切后产生大小不同的片段,因而可以达到酶切分型的目的。经敏感性试验表明,该方法可以检出10~0个细菌。     

副溶血性弧菌O抗原的分子分型研究

为建立一种针对副溶血性弧菌O抗原进行分子分型的方法,以实验室前期从水产品中分离的6个O群的54株副溶血性弧菌为研究菌株,采用长聚合酶链式反应扩增副溶血性弧菌O抗原合成相关基因,使用3种限制性内切酶(Eco R V,BsmⅠ和XmnⅠ)对扩增产物进行限制性酶切。经琼脂糖凝胶电泳,NTSYS软件进行聚类分析,结果表明:琼脂糖凝胶电泳和聚类分析均可得到清晰的电泳图和准确的分型。聚类结果显示:经Eco R V、Bsm I和Xmn I 3种限制性内切酶酶切,分别在相似系数为0.72,0.78,0.86时将54株菌分成6个群,且分型结果同副溶血性弧菌的O群血清分型完全吻合。所建立的分子分型方法具有良好的分辨力,产生的条带明亮、清晰,方法操作简便,试验成本低。     

PCR-RFLP方法检测和鉴别五种李斯特菌

目的 建立快速检测和鉴别单增李斯特菌、绵羊李斯特菌、英诺克李斯特菌、威尔李斯特菌和格氏李斯特菌等常见李斯特菌的方法。方法 采用PCR-RFLP技术,首先通过引物“Lis1A-Lis1B”对李斯特菌属iap基因进行扩增,扩增产物大小约1.4 kb,然后用限制性内切酶DdeⅠ对PCR产物进行酶切,电泳观察具有种间特异性的酶切谱带进行鉴别。结果 上述5种李斯特菌的PCR扩增产物经内切酶DdeⅠ消化后,得到片段大小不同的,具有种间差异的特异性酶切图谱。结论 本实验建立的PCR-RFLP方法可以用于上述5种常见李斯特菌的快速检测和鉴定。     

不同来源的霍乱弧菌毒力特征的质谱学分析

目的: 分析本地区食品风险监测样品中霍乱弧菌的分布,评价使用VITEK-MS微生物质谱进行霍乱弧菌快速鉴定的优劣。方法: 从淡水产品和病人分离获得霍乱弧菌,分别进行VITEK-2生化鉴定和VITEK-MS质谱鉴定。血清凝集和胶体金显色法检测霍乱弧菌血清型,PCR检测ctx毒力基因。通过全基因组测序分析霍乱弧菌毒力基因类型。结果: 经快速鉴定检出23株霍乱弧菌,17株为非O1/O139血清型,6株为O139型并且携带ctx毒力基因。VTIEK-MS微生物质谱检出的霍乱弧菌具有典型的指纹图谱特征,O139型霍乱弧菌具有典型的特有峰和缺失峰特征。全基因组测序显示O139血清型具ctxAB、ace和tcp毒力岛等毒力基因,而非O1/O139株则只具有辅助毒力因子,个别腹泻病例株携带了zot基因。结论: 非O1/O139和O139型霍乱弧菌无论在毒力基因种类和质谱特征上都具有显著的差异,广泛应用VITEK-MS微生物快速鉴定技术进行霍乱弧菌血清鉴定有望成为霍乱监测的新助力。     

2020—2021年绍兴市水产品和腹泻患者来源的霍乱弧菌耐药性与分子特征分析

目的 了解2020—2021年绍兴市霍乱弧菌的血清型、霍乱毒素编码基因（ctxAB）携带率、耐药性及分子分型情况。方法 收集2020—2021年分离自水产品、腹泻患者粪便的霍乱弧菌68株,使用玻片凝集法进行血清分型;利用实时荧光PCR检测ctxAB基因;采用微量肉汤稀释法和脉冲场凝胶电泳（PFGE）分别进行药物敏感试验和分子分型。结果 68株霍乱弧菌只有3株为O1群小川型,其余65株均为非O1/O139血清群;所有菌株均未检出ctxAB基因;菌株对头孢他啶、头孢他啶/阿维巴坦和复方新诺明的耐药率分别为45.6%、38.2%和36.8%,对四环素、替加环素、阿奇霉素、阿米卡星敏感,17株菌对3种及以上抗生素耐药;菌株经Not I酶切后的PFGE图谱呈多样性,水产品分离株与病例分离株的分子分型差别较大。结论 2020—2021年绍兴市霍乱弧菌主要为非O1/O139血清群,不携带毒力基因ctxAB,对多种抗生素耐药,分子特征复杂多样。     

利用线粒体DNA Cyt b基因PCR-RFLP分析方法鉴别羊肉和鸭肉

建立了一种利用线粒体DNA（mtDNA） Cyt b基因PCR-RFLP分析来鉴别羊肉和鸭肉的方法。采用一对通用引物扩增绵羊、山羊和鸭的mtDNACytb基因,并对扩增产物用DNA限制性内切酶Bsu36I和SpeI进行酶切,电泳分析酶切产物的变化。结果表明通用引物可扩增羊和鸭472bp的PCR产物,经两种内切酶酶切后,绵羊、山羊和鸭的PCR产物分别被切为大小不同的片段,其中绵羊和山羊被SpeI切为213bp和259bp,而鸭则被Bsu36I切为95bp和377bp。利用PCR-RFLP分析mtDNA Cyt b基因的方法操作简单,是一种快速鉴别羊肉和鸭肉的可靠方法。      

茶树叶绿体DNA的PCR-RFLP反应体系优化

通过正交设计对影响茶树聚合酶链反应-限制性片段长度多态性(PCR-RFLP)反应体系的主要因素进行优化,快速确立适合茶树叶绿体DNA PCR-RFLP分析的扩增体系和酶切体系。结果表明：最佳PCR扩增体系为100ng模板DNA、200μmol/L dNTPs、1.5mmol/L MgCl2、50ng叶绿体引物、3U TaqDNA聚合酶、加ddH2O至25μL;最佳酶切体系为6μL扩增产物用量、2U限制性内切酶量、1×限制性内切酶buffer、加ddH2O至15μL,37℃酶切6h。利用优化的反应体系,对30个茶树品种叶绿体DNA进行PCR-RFLP扩增,可获得清晰扩增图谱和多态性酶切图谱。     

利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism,T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记,从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记,上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记,下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳,片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼,而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱,能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同,结果具有可重复性。没有出现物种内多态的现象。大多数情况下,实际得到的末端片段长度与理论值非常接近,只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确,适用于鉴定动物种类。     

进口黑虎虾中霍乱弧菌的鉴定及其挥发性产物分析

目的对进口黑虎虾中分离的菌株F14-2进行种属鉴定,通过顶空气相色谱-质谱联用法(headspace gas chromatography-mass spectrometry,HS-GC-MS)分析分离菌株液态培养代谢的挥发性产物。方法应用VITEK 2 Compact全自动微生物鉴定系统分析菌株的生理生化特征,利用荧光定量PCR特异性扩增检测病原菌,通过顶空气相色谱-质谱联用法分析分离菌株F14-2的代谢的挥发性产物。结果菌株F14-2为革兰氏阴性菌,生理生化特征与霍乱弧菌(Vibrio cholerae)的相似性为98%,荧光定量PCR检测和血清凝集试验进一步确认为非O1非O139群霍乱弧菌,菌株F14-2在营养肉汤(nutrient broth)中代谢的挥发性产物主要有乙醇、异戊醇、乙酸和2-乙基己醇。结论进口黑虎虾中分离的菌株F14-2鉴定为霍乱弧菌,该菌产生的挥发性产物为霍乱弧菌的鉴定提供参考。     

中国葡萄酒产区酒酒球菌种质资源遗传多样性分析

为了解我国葡萄酒产区酒酒球菌种质资源遗传多样性,对22 株筛选自我国不同葡萄酒产区的乳酸细菌进行了种特异性聚合酶链式反应（species-specific polymerase chain reaction,PCR）分析、16S rRNA序列分析和扩增片段长度多态性分析（amplified fragment length polymorphism,AFLP）基因分型。种特异性PCR和16S rRNA序列分析表明22 株分离株为酒酒球菌（Oenococcus oeni）。建立了O. oeni基于HindⅢ和MseⅠ为内切酶的AFLP分析体系,对16 对引物组合进行了筛选,结果表明HT-MA、HT-MT、HT-MC、HG-MA、HG-MT、HC-MT为O. oeni AFLP分析的最佳引物组合,且实验重复性在98%以上。对O. oeni的AFLP分析结果显示：22 株O. oeni分为3 个簇群,且簇群间遗传相似性系数较小。所以,以HindⅢ和MseⅠ为内切酶的AFLP技术是研究O. oeni基因分型的有效方法,我国葡萄酒产区的O. oeni具有丰富的遗传多样性,O. oeni菌株间的遗传相似性系数不仅仅与其生态地理分布有关,可能还与其所处的微生态和其他因素有关。     

Tracking verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 in Irish cattle

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge.     

Polyester Cloth-Based Hybridization Array System for Identification of Enterohemorrhagic Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli (EHEC-7) serogroups targeted by U. S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100 % inclusivity and 100 % exclusivity characteristics with respect to detection of the various markers among 89 different E. coli strains, with various marker gene profiles and 15 different strains of non-E. coli bacteria.     

Microbial respiration and diffusive transport of O2, 16O2, and 18O15O in unsaturated soils and geologic sediments

Molecular oxygen (O2) in unsaturated geologic sediments plays an important role in soil respiration, biodegradation of organic contaminants, metal oxidation, and global oxygen and carbon cycling, yet little is known about oxygen isotope fractionation during the consumption and transport of O2 in unsaturated zones. We used a laboratory kinetic cell technique to quantify isotope fractionation due to respiration and a numerical model to quantify both consumptive and diffusive fractionation of O2 isotopes at a field site comprised of unsaturated lacustrine sandy materials. The combined use of laboratory-based kinetic cell experiments and field-based isotope transport modeling provided an effective tool to characterize microbial respiration in unsaturated media. Based on results from the closed-system kinetic cells, O2 consumption and isotope fractionation were attributed to the alternative cyanide-resistant respiration pathway. At the field site, the modeled depth profiles for O2 and delta18O matched the measured in situ data and confirmed that the consumption of O2 was via the alternative respiration pathway. If the cyanide-resistant respiration pathway is indeed widespread in soils, its high oxygen isotope enrichment factor could help to explain the discrepancy between the predicted present-day Dole effect (+20.8/1000) and the observed Dole effect (+23.5/1000). Thus, further soil O2 isotope studies are needed to better characterize and model the fractionation of oxygen isotopes during subsurface respiration and the potential impact on the isotopic content of atmospheric O2.     

Latex agglutination assays for detection of non-O157 Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145

Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 μm l of latex-IgG reagent containing 2.0 to 2.8 μm g IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.     

A rapid procedure for the detection and isolation of enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, O145 and O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables

Human infections with Enterohaemorrhagic Escherichia coli strains (EHEC) as agents of Haemorrhagic Colitis (HC) and Haemolytic Uraemic Syndrome (HUS) are frequently associated with the consumption of EHEC contaminated foodstuffs of different origins. EHEC O26, O103, O111, O118, O121, O145 and O157 strains are responsible for the majority of HC and HUS cases worldwide. In May 2011, the emerging aggregative EHEC O104:H4 strain caused a large outbreak with high HUS incidence in northern Germany. Contaminated sprouted seeds were suspected to be the vehicles of transmission. The examination of vegetables retailed for raw consumption revealed low numbers of E. coli (< 100 cfu/ g) together with high titres of Enterobacteriaceae and Pseudomonas (approx. 5.6 × 10⁷ cfu/ g). Specific methods of EHEC detection adapted to vegetables are not yet published. Therefore, we have developed a rapid and sensitive method for detecting low EHEC contamination in vegetables (1–10 cfu/25 g) with artificially EHEC contaminated ready-to-eat salads. A 6-hour enrichment period in BRILA-broth was sufficient to detect 1–10 EHEC from spiked samples after plating 0.1 ml portions of enrichment culture on selective TBX-agar and CHROMagar STEC plates that were incubated at 44 °C overnight. Unlike EHEC strains, the growth of bacteria of the plant flora was substantially inhibited at 44 °C. DNA for real-time PCR detection of EHEC characteristic genes (stx₁, stx₂, eae, ehxA, and O-antigen associated) was prepared with bacteria grown on TBX-agar plates. The storage of EHEC inoculated salad samples for 72 h at 6 °C resulted in a significant reduction (mean value 14.6%) of detectable EHEC, suggesting interference of EHEC with the resident plant microflora. CHROMagar STEC was evaluated as a selective medium for the detection of EHEC strains. Growth on CHROMagar STEC was closely associated with EHEC O26:[H11], O111:[H8], O118:H16, O121:[H19], O145:[H28], O157:[H7] and aggregative EHEC O104:H4 strains and with the presence of the terB gene (tellurite resistance). TerB sequences were found in 87.2% of 235 EHEC but only in only 12.5% of 567 non-EHEC strains. EHEC strains which did not grow on CHROMagar STEC were negative for terB as frequently observed with EHEC O103:H2 (52.9%) and sorbitol-fermenting O157:NM strains (100%). The enrichment and detection method was applied in the examination of sprouted seeds incriminated as vehicles in the EHEC O104:H4 outbreak in Germany. Aggregative EHEC O104:H4 could be detected and isolated from a sample of sprouted seeds which was suspected as vector of transmission of EHEC O104 to humans.     

Isolation of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Ground Beef Using Modified Rainbow Agar and Post-Immunomagnetic Separation Acid Treatment

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.     

Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 experimentally inoculated in beef carcass swabs, beef trim, and ground beef

Numerous foodborne outbreaks are attributed to Shiga toxin-producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 10(2), and 10(3) CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 10(2), and 10(3) CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 10(2) CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STEC in irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.     

Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food

The aim of the current study was to evaluate a multiplex PCR (mPCR) detection test combined with the evaluation of a previously described isolation method.Minced beef, raw-milk cheese and sprouted seed samples were inoculated with low amounts (7-58 cfu 25 g⁻¹) of non-stressed, cold-stressed or freeze-stressed clinical STEC strains, including serogroups O26, O103, O111, O145, sorbitol fermenting (SF) O157 and non-sorbitol fermenting (NSF) O157. The inoculated pathogen was detected using a 24 h-enrichment followed by an mPCR protocol, and in parallel isolated using an enrichment step of 6 and 24 h, followed by selective plating of the enriched broth and selective plating of the immunomagnetic separation (IMS) product. Recovery results were evaluated and compared.Successful mPCR detection and isolation was obtained for non-stressed and cold-stressed STEC cells in minced beef and raw-milk cheese samples, except for serogroups O111 and SF O157. For freeze-stressed cells and sprouted seed samples, false negatives were often found. Isolation was better after 24 h-enrichment compared to 6 h-enrichment. IMS improved in some cases the isolation of non-stressed and cold-stressed cells belonging to serogroups O111 and O157 from minced beef and raw-milk cheese and freeze-stressed cells of all tested serogroups from minced beef.     

Transfer of verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 from fleece to carcass during sheep slaughter in an Irish export abattoir

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from fleece to dressed carcasses of 500 sheep, and to establish the virulence potential of recovered VTEC. Individual sheep were tracked and sampled (10 g fleece, full carcass swab) through the slaughter process. Samples were examined for the presence of verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay and positive samples were further screened for the presence of the above five serogroups by real-time PCR. VTEC cells were recovered from PCR positive samples by serogroup specific immunomagnetic separation and confirmed by serogroup specific latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR and isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). VTEC O26 was recovered from 5/500 (1.0%) fleece and 2/500 (0.4%) carcass samples. VTEC O157 was isolated from 4/500 (0.8%) fleece samples and 3/500 (0.6%) carcass samples. E. coli O103 was recovered from 84/500 (16.8%) fleece and 68/500 (13.6%) carcasses, but only one E. coli O103 isolate (0.2%) carried vt genes. E. coli O145 was recovered from one fleece sample, but did not carry vt genes. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from fleece to carcass was not observed with PFGE showing that VTEC O26 isolates from a matched fleece/carcass “pair” were not identical. This study shows that while VTEC O157 are being carried by sheep presented for slaughter in Ireland, other potentially clinically significant verotoxin producing strains (particularly VTEC O26) are emerging.     

大肠埃希氏菌O157和O111能力验证样品的研制

目的研制植物源性成分绿豆基质中O157和O111检测能力验证样品,建立有效的样品制备流程来考核实验室检测致泻大肠埃希氏菌O157和O111的能力,根据参加实验室检测结果是否准确及可靠来评价实验室检测水平,并促进实验室检测体系进一步完善。方法选用O157和O111的ATCC标准菌株制备样品,以绿豆粉为基质,真空冷冻干燥技术制备样品。采用显色培养基、荧光定量PCR、全自动细菌生化鉴定仪、VIDAS全自动免疫荧光分析仪和血清型鉴定进行定性检测,并进行菌落计数。结果随机选取每一批阳性样品中的30个样品,每个样品中目标菌数在21~60 CFU之间,并且A组与B组阳性样品中均检测出目标菌,而阴性样品中均没有出现目标菌。结论表明采用本方法制备的能力验证样品均一稳定,能较好地评价实验室致泻大肠埃希氏菌O157和O111的检测能力。