空肠弯曲菌、单增李斯特菌和大肠杆菌O157多重PCR分子检测研究

目的建立同时检测空肠弯曲菌、单核细胞增生性李斯特菌(简称单增李斯特菌)和大肠杆菌O157的多重聚合酶链反应(PCR)检测技术。方法根椐空肠弯曲菌mapA基因序列、单增李斯特菌的编码溶血素(hly)基因序列和大肠杆菌的O157抗原(rfbE)基因序列,分别设计了3对特异引物,PCR扩增的目的基因片段为589 bp、360 bp和194bp;并对反应条件进行了优化。结果对3种致病菌检测,多重PCR检测DNA的敏感度分别为空肠弯曲菌2.264 ng、单增李斯特菌37.92 ng、大肠杆菌2.100 ng,样品检测时间6~8 h。结论该方法操作简便,检测周期短,特异性和敏感度高,为同时检测污染样品中空肠弯曲菌、单增李斯特菌和大肠杆菌O157奠定了基础。     

多重PCR结合基因芯片技术检测11种致病菌方法的建立

目的建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法。方法筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的基因。设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有11种特异探针的基因芯片杂交。用扫描仪扫描,判定细菌种类。结果该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng。结论多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性。     

多重实时荧光PCR快速检测沙门菌和单增李斯特菌

目的 建立可同时检测沙门菌、单增李斯特菌的双重实时荧光PCR快速检测方法,并应用于食品样品的检测.方法 根据GenBank上下载的沙门菌invA基因、单增李斯特菌hlyA基因的保守区序列分别设计特异性引物和TaqMan探针,建立并优化双重实时荧光PCR反应体系.对该方法的特异性、敏感性和稳定性进行评估,并应用于食品标本...     

2008年武汉市食品中食源性致病菌污染状况分析

[目的]了解武汉市食源性致病菌在食品中的污染状况,提高食品卫生监测和危险性评估能力。[方法]2008年采集8大类食品,共计218份样品按照国家食源性疾病监测网2008年工作手册提供的检测方法进行沙门菌、金黄色葡萄球菌、副溶血性弧菌、单增李斯特菌、大肠杆菌O157：H7、弯曲菌和阪崎肠杆菌7种食源性致病菌的检测。[结果]于2008年218份样品中共检出致病菌15株,总检出率为6．9％（15／218）。其中沙门菌检出5株,检出率2．5％为（5／198）;副溶血性弧菌检出3株,检出率为15％（3／20）;空肠弯曲菌检出2株,检出率为11．1％（2／18）;金黄色葡萄球菌检出5株,检出率为11．1％（5／45）。样品中未检出单增李斯特菌、大肠杆菌0157：H7和阪崎肠杆菌。生畜肉、生禽肉、动物性水产品、即食食品、蔬菜沙拉和面米食品的污染率分别为20％（1／5）,16．6％（3／18）,17．6％（3／17）,2．7％（2／73）,3．3％（1／30）,20％（5／25）。[结论]武汉市食品中存在食源性致病菌的污染,生畜肉、生禽肉、动物性水产品和面米食品受到的污染最为严重。沙门菌、副溶血性弧菌、金黄色葡萄球菌是武汉市食源性疾病发生的危险因素。     

基于双启动寡聚核苷酸引物的沙门菌、志贺菌、弯曲杆菌、耶尔森菌多重实时荧光PCR方法的建立

目的建立多重实时荧光PCR同时检测沙门菌、志贺菌、弯曲杆菌、耶尔森菌的方法。方法通过设计针对沙门菌、志贺菌、弯曲杆菌和耶尔森菌的双寡聚核苷酸引物,来建立这4种病原体的多重PCR检测体系。结果建立的多重PCR体系可同时特异性扩增4种病原体的DNA片段。沙门菌和弯曲杆菌的特异性和敏感度都是100.0%,志贺菌和耶尔森菌的敏感度是100.0%,特异性分别是99.3%和98.6%。最低检测限均为103 copies/mL。结论该体系可快速、有效、准确检测出样品中4种病原体的存在,可用于肠胃道相关病原体感染的辅助评价。     

模拟粪便标本中的单增李斯特菌的分离和检测

目的 建立针对粪便标本中单增李斯特菌的分离鉴定方法,评价方法的检测下限,以提高感染人群标本中单增李斯特菌的检出率,了解我国人群中该菌的携带及感染情况。 方法 对模拟人粪便标本进行二次增菌,采用Real-time PCR检测,并同时分离培养获得该病原菌。 结果 当每克模拟粪便标本中含有7 cfu的单增李斯特菌时,经过增菌后的标本用Real-time PCR方法可检测出阳性结果,并能够通过单增李斯特菌的选择培养基分离得到病原菌。 结论 本研究为从粪便标本中分离单增李斯特菌和由单增李斯特菌引起的食物中毒事件的病原学调查提供了技术支持,有利于对我国人群中单增李斯特菌的携带或感染状况进行调查分析。     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应（PCR）方法。方法本研究依据沙门菌侵袭基因正调节蛋白（hilA）基因、变形杆菌溶血素（hpmA）基因和金黄色葡萄球菌特异性pSa-442序列,运用PrimerPremier5．0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401bp、256bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16（4^3）正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为：94．07pg沙门菌DNA,140．85ng变形杆菌DNA,1．41ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4h培养后样品的最低检测限度分别为：沙门菌10^0菌落形成单位（CFU）／mL、变形杆菌10^1CFU／mL、金黄色葡萄球菌10^0CFU／mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

模拟粪便标本单增李斯特菌和伊氏李斯特菌TaqMan双重实时荧光定量-聚合酶链反应检测方法

目的建立一种TaqMan双重实时荧光定量-聚合酶链反应(real-time PCR),用于模拟粪便标本中单增李斯特菌和伊氏李斯特菌的快速检测。方法以单增李斯特菌特异基因hly和伊氏李斯特菌特异基因smcL作为靶基因,合成2对引物和其相应的荧光探针,制作标准曲线,建立从模拟粪便标本中直接检测单增李斯特菌和伊氏李斯特菌的TaqMan双重real-time PCR。利用李斯特菌属中其他种李斯特菌及常见致病菌验证引物、探针的特异性;通过制备模拟粪便标本并对其进行二次增菌后,分别提取DNA,采用TaqMan双重real-time PCR进行检测,以达到快速检测单增李斯特菌和伊氏李斯特菌的目的。结果采用本研究建立的TaqMan双重real-time PCR对模拟粪便标本的检测结果显示特异性良好,与其他种李斯特菌和其他病原菌均无交叉反应。模拟粪便标本中单增李斯特菌和伊氏李斯特菌的检测下限分别为2.45×103cfu/g和2.92×103cfu/g;模拟粪便标本在3 h内可得出检测结果,当模拟粪便标本中单增李斯特菌含量为6 cfu/g和伊氏李斯特菌含量为5 cfu/g时,经过增菌后使用双重real-time PCR可检测出阳性结果。结论本研究建立了以单增李斯特菌的特异基因hly和伊氏李斯特菌的特异基因smcL为靶基因的TaqMan双重realtime PCR检测方法,该方法具有特异性好、敏感性高、快速易操作等优点,可用于临床、食品及环境标本的快速诊断,以及我国人群中单增李斯特菌和伊氏李斯特菌的携带或感染状况的调查分析。     

沙门菌、志贺菌和副溶血性弧菌的多重PCR快速检测技术的建立与应用

目的建立快速同时检测沙门菌、志贺菌和副溶血弧菌的多重聚合酶链反应（PCR）技术。方法根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌tdh基因序列设计引物，进行PCR扩增及电泳检测。同时优化反应体系，测定特异性和灵敏度。结果该多重PCR技术能在8h内同时检测3种目的菌，通过检测其他9种常见食源性致病菌，结果表明该方法特异性好，多重PCR检测灵敏度分别为：沙门菌10^4cfu／mL，志贺菌10^2cfu／mL，副溶血性弧菌10^4cfu／mL。并进行了人工模拟食品和粪便样品的检测。结论初步建立能在8h内快速、灵敏、特异地同时测定沙门菌、志贺菌和副溶血性弧菌的多重PCR检测技术。     

检测胎儿弯曲菌三种亚种的多重PCR方法建立及初步应用

目的 建立一种检测胎儿弯曲菌胎儿亚种、性病亚种和龟亚种的多重PCR检测方法。方法 使用针对胎儿弯曲菌、性病亚种和龟亚种的特异性引物,优化反应体系及反应条件,使用38株菌株（18株胎儿弯曲菌和20株非胎儿弯曲菌）验证其特异性,并将该方法应用于53份爬行动物粪便筛查。结果 针对3种亚种均可扩增相应的片段,胎儿亚种只有1条359 bp大小的条带、性病亚种有2条分别为359 bp和156 bp大小的条带,龟亚种有2条分别为359 bp和266 bp大小的条带。优化后的最佳扩增条件：退火温度58℃,引物MG3f和Cf359r浓度为0.1μmol/L,ISC2mf和ISC2mr、CoA266f和CoA266r的浓度0.2μmol/L。对胎儿亚种、性病亚种和龟亚种的检出限分别为0.40 ng/μL、0.39 ng/μL和0.47 ng/μL。使用18株胎儿弯曲菌和20株非胎儿弯曲菌其特异性为100%。对53份爬行动物粪便进行筛查中有1份龟亚种阳性并分离出了相应的菌株。结论 本研究建立的多重PCR方法能利用1个反应体系同时鉴别3种亚种,从而为菌株快速鉴定、流行病学调查和溯源研究提供技术支持。     

Development of a triplex PCR assay for the specific detection of Campylobacter jejuni,Salmonella spp., and Escherichia coli O157:H7

A triplex PCR assay was developed and evaluated for efficacy in detecting Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7 in a variety of raw and ready-to-eat food products. Following a short enrichment period, artificially contaminated food samples were subjected to a triplex PCR assay, which incorporated published primers for each food pathogen, a protocol for sample collection, and a PCR procedure designed specifically for the assay. The selected primers amplified fragment sizes of 159 bp, 252 bp, and 360 bp for C. jejuni, E. coli O157:H7, and Salmonella spp., respectively. This assay provides specific and reliable results and allows for the cost-effective detection of all three bacterial pathogens in one reaction tube.     

Bactericidal activities of milk lipids

The bactericidal capacity of digestion products of bovine milk triglycerides and membrane lipids was tested in vitro using Escherichia coli O157:H7, Salmonella enteritidis, Campylobacter jejuni, Listeria monocytogenes, and Clostridium perfringens. C10:0 and C12:0 fatty acids and digestion products of sphingolipids appeared to be effective bactericidal agents, whereas digestion products of phosphoglycerides were moderately bactericidal. Thus, milk fat sphingolipids and triglycerides, particularly those containing C10:0 and C12:0 fatty acids, may protect against food-borne gastroenteritis.     

Development of a ceuE-based multiplex polymerase chain reaction (PCR) assay for direct detection and differentiation of Campylobacter jejuni and Campylobacter coli in Thailand.

A novel ceuE-based multiplex PCR system was developed as an efficient diagnostics test to detect and differentiate C. jejuni and C. coli. There is no cross reactivity between C. jejuni and C. coli. In addition, the assay does not produce a positive signal from other enteric bacteria including Salmonella, Shigella and Escherichia coli strains. Campylobacter detection sensitivity was determined to be equivalent to previously reported PCR for other enteric bacteria. We also noticed that silicon dioxide extraction can improve Campylobacter detection sensitivity from infected stool samples. It was demonstrated that the PCR assay developed in this study had a much better Campylobacter detection rate than the traditional culturing method (77% versus 56%). However, we also identified small numbers of culture positive stools (8%, or 16 out of 202 samples) that did not yield PCR positive results for Campylobacter. These PCR negative/culture positive stools were proven to be inhibitory to PCR amplification.     

Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E. coli.

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7 Copyright 1999 Academic Press.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the gamma-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.