简介:ThegenomeoftheenterohemorrhagicEscherichiacoliO157:H7EDL933contains177“O”-islands(OIs).TostudytheirpotentialcontributiontotheO157-specificpathogenicity,wesurveyedthedistributionof22OIsbyPCRandDNAhybridizationin17isolatesofShigatoxinproducing(Stx-positive)E.coliO157:H7,andcomparedwiththeirdistributionin21isolatesofStx-negativeE.coliO157and21isolatesofnon-O157entericpathogens.Fourteenof22OIswerepresentinnon-O157entericpathogensanalyzed.Eightof22OIswerefoundonlyinthe17Shigatoxin-(Stx)positiveE.coliO157:H7isolates,buttheywereabsentfromthe21Stx-negativeE.coliO157:NMandO157Hundisolatestested.Amongthe8OIs,onlyOI43orOI48wereexclusivelydetectedinStx-positiveE.coliO157:H7,absentfromneitherofStx-negativeE.coliO157andnon-O157entericpathogens,suchasSalmonella,ShigeUa,Citrobacter,Vibriocholera,enteropathogen-icE.coli(EPEC),enteroadherentE.coli(EAEC),enteroinvasiveE.coli(E1EC)andenterotoxingenicE.coli(ETEC).TheOI43andOI48are83kbinsizeandidenticalinDNAsequences,whichencodegenesforurease,telluriteresistanceandadherence.ByanalyzingtheirjunctiongeneswithPCRandDNAhybridization,wefoundthat21ChineseisolateshaveOI48only.However,for7Japanesepatientisolates,4haveOI43and3haveOI48;forAmericanisolates,2havebothofO143andOI48,2haveOI48only.ThesedataconfirmedthehighlyplasticityofthepathogenicE.coligenome.TheuniquepresenceofOI43/OI48inStx-positiveE.coli0157:H7denotesitscriticalroleinthepathogenicityspecifictothispathogen.
简介:目的:利用Red系统构建肠出血性大肠杆菌O157:H7的sRNA基因E40缺失突变株。方法:选取本实验室预测并经过实验验证的sRNA基因,根据NCBI上相应的序列,设计2对引物分别扩增该sRNA基因的上下游分别长464和455bp的同源臂,经PCR扩增,构建到相应的载体,最后以构建好的含上下游同源臂和卡那霉素抗性基因的长约2500bp的线性片段作为打靶片段,在Red重组系统的作用下与sRNA基因E40的上下游同源区域发生同组,从而把sRNA基因从基因组上置换下来,之后利用质粒pCP20将FRT位点间的卡那霉素抗性基因消除。结果与结论:构建了出血性大肠杆菌O157:H7的sRNA基因E40的缺失突变株,为进一步研究sRNA基因在出血性大肠杆菌O157:H7生长及致病过程中所起的功能奠定了良好的基础。
简介:ToconstructandexpressthefusionproteinStx2B-IntiminC300ofEHEC0157:H7,andtofurtherinvestigateitsimmunoprophyiacticpotential,thegeneofStx2B(stx2b)fromEHEC0157:H7chromosomewasclonedintopMD18-Tvector.Thereafter,theamplifiedgenewasclonedintoprokaryoticexpressionplasmidpET-28a(+)-eaeC300,whichwasconstructedpreviously.TherecombinantpasmidpET-28a(+)-stx2b-eaeC300wastransformedintoE.coliBL21(DE3).Afterinducement,theproteinStx2B-IntiminC300wassuccessfullyexpressedandanalyzedwithsodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE),WesternblottingandN-terminalaminoacidresidualsequencing.Toevaluateitsimmunoprophyiacticpotential,itwasprimarilypurifiedbyion-exchangechromatographyandinjectedinto30BALB/cmicewithAl(OH)3inthesubscapularregion.Tendaysafterthelastboostervaccination,20micewereattackedwithEHEC0157:H7lysateandtheprotectiveefficacywasobserved.Inthepresentstudy,thegeneofStx2B-IntiminC300wassuccessfullyclonedintopET-28a(+)vector.TheresultsofSDS-PAGEandWesternblottingassayshowedthatthefusionproteinwassuccessfullyexpressedintheinclusionbodyform,accountingfor25%oftotalexpressionproducts,anditsmolecularweightwasabout43kDa.TheresultoftheN-terminalaminoacidresidualsequencingshowedthatitwasidenticaltothatofthemoleculardesigned.Thepuritywasabout75%afterprimarypurification.AnimaltestsrevealedthatthefusionproteinStx2B-IntiminC300haselicitedhightiterofprotectiveantibodyrelatively.TheseresultsdemonstratethatthefusionproteinStx2B-IntiminC300issuccessfullyexpressedinprokaryoticexpressionsystemandshowscertainimmunoprophyiacticpotential.
简介:应用压电免疫传感器快速检测了大肠杆菌O157:H7(E.coliO157:H7).将巯基乙酸(MACA)自组装在AT切向的压电金电极上,以N-羟基琥珀酰亚胺(NHS)为反应介质,形成酰氨键固定大肠杆菌抗体.并通过α-甲基丙烯酸(MAA)把抗体修饰在纳米磁球上.采用夹心法将抗原夹在两个抗体间,压电石英晶体交流阻抗(PQCI)和循环伏安法对修饰过程进行了表征,抗原浓度与形成夹心复合物前后的PQCI振动频移相关.其免疲传感器能在3h内检测2×10^3~8×10^8CFU/mL范围内的目标细菌,10^6CFU/mL情况下重复性实验的RSD在7%以下。
简介:摘要肠出血大肠杆菌O157H7作为一种新型病原菌,这是当前世界上食源性疾病的重要诱因,由其所引发的各种感染问题,严重威胁着人类的生命健康,并且还会引发各种并发症。那么在这样的背景下,从分子生物学的角度出发,对肠出血大肠杆菌O157H7进行相应的检测调查,能够为肠出血大肠杆菌O157H7疾病的预防和诊断提供有效的支撑杆。因此本文就对肠出血大肠杆菌O157H7的分子生物学检测方法研究进展进行有效的分析和探究。
简介:目的了解我国食品中分离的110株大肠埃希菌O157的耐药及脉冲场凝胶电泳(PFGE)分子分型特征,完善我国食品中大肠埃希菌O157菌株特征的基础信息,为该菌的风险评估提供依据。方法使用琼脂稀释法对确认的110株大肠埃希菌O157进行药敏试验,完成耐药特征的分析。参照美国疾病预防控制中心PulseNet试验方法,对110株大肠埃希菌O157,运用XbaⅠ酶进行酶切并完成PFGE分析,利用BioNumerics软件对分离株的指纹图谱进行聚类分析。结果1110株菌中,43株菌至少对一种抗生素有抗性。耐药率最多的前三种抗生素依次是四环素(30.0%,33/110),磺胺甲恶唑(29.1%,32/110),萘啶酸(26.4%,29/110);2一共有24个耐药谱,耐两种以上抗生素的菌株有34株,耐3种以上抗生素的多重耐药菌株有32株。最常见的三种耐药谱依次是SMX(6),AMP-NAL-SMX-SXT-TET(6),AMP-CHL-NAL-SMX-SXT-TET(4)/AMP-SMX-SXT-TET(4)/TET(4);3大肠埃希菌O157非H7(O157∶hund)对所测试的抗生素的耐药率明显高于大肠埃希菌O157∶H7(χ2=72.010P〈0.05)。其中37株携带了志贺毒素基因的大肠埃希菌O157∶H7仅对磺胺甲恶唑(2.7%,1/37)、萘碇酸(2.7%,1/37)有耐药,没有多重耐药菌株;4通过不同种类食品中大肠埃希菌O157菌株耐药率比较发现,从生猪肉、生禽肉中分离的菌株耐药率相对高于其他食品种类;5PFGE分子分型研究显示菌株具有基因多态性,且可以很好将大肠埃希菌O157非H7和大肠埃希菌O157∶H7菌株区分开。结论我国食品中分离的大肠埃希菌O157耐药现象严重。我们应加强养殖环节和零售环节食源性致病菌,特别是大肠埃希菌O157(包括产志贺毒素大肠埃希菌O157)菌株药敏特征的监测,探明食品与养殖环节菌株耐药的传播关系,并为国家制定科学的养殖业抗生素用药提供依据。
简介:AbstractWe conducted environmental surveillance to detect avian influenza viruses circulating at live poultry markets (LPMs) and poultry farms in Guangxi Autonomous Region, China, where near the China-Vietnam border. From November through April 2017-2018 and 2018-2019, we collected environmental samples from 14 LPMs, 4 poultry farms, and 5 households with backyard poultry in two counties of Guangxi and tested for avian influenza A, H5, H7, and H9 by real-time reverse transcription-polymerase chain reaction (rRT-PCR). In addition, we conducted four cross-sectional questionnaire surveys among stall owners on biosecurity practices in LPMs of two study sites. Among 16,713 environmental specimens collected and tested, the median weekly positive rate for avian influenza A was 53.6% (range = 33.5% - 66.0%), including 25.2% for H9, 4.9% for H5, and 21.2% for other avian influenza viruses A subtypes, whereas a total of two H7 positive samples were detected. Among the 189 LPM stalls investigated, most stall owners (73.0%) sold chickens and ducks. Therefore, continued surveillance of the avian influenza virus is necessary for detecting and responding to emerging trends in avian influenza virus epidemiology.
简介:目的调查一起食物中毒事件,分离并鉴定其致病微生物是否为大肠埃希菌O157。方法根据患者临床表现和标本种类,按照临床微生物检验法规,利用小型全自动荧光酶免疫分析仪(mini-VIDAS)、全自动细菌鉴定药敏分析系统、全自动微生物鉴定仪(VITEK32),分别进行初筛和系统生化与药敏鉴定,并结合血清学试验作出最终鉴定。结果该起食物中毒的病原微生物被mini-VIDAS初筛为大肠埃希菌O157阳性菌株,后经系统鉴定为产毒型大肠埃希菌O8:K40,K47血清型。9份标本中有6份检出产毒型大肠埃希菌O8:K40,K47血清型,检出率为66.7%(6/9),其中食品1份,呕吐物3份,肛拭2份,所有标本均未检出金黄色葡萄球菌、蜡样芽胞杆菌、霍乱弧菌、副溶血性弧菌、志贺菌、沙门菌及其他致泻性大肠埃希菌。结论该起食物中毒由产毒型大肠埃希菌O8:K40,K47血清型引起。产毒型大肠埃希菌(O8:K40,K47血清型)标本经mini-VIDAS初筛可以产生假阳性结果,需进一步确证。
简介:一汽集团积极响应国家战略和法规要求,成功获批工信部新能源车开发项目,重点研发推广离合器耦合式电机总成(CCM)。离合器耦合式电机总成为“P2”构型,该构型技术门槛高、开发难度大,离合器耦合式电机总成集成双质量飞轮、分离离合器和驱动电机,集成化程度高,结构紧凑,其中双质量飞轮和分离离合器为二次开发,驱动电机总成为自主开发。CCM已成功搭载红旗H7PHEV车型,并于2016年投放市场,市场反馈良好。