基于实时荧光环介导等温扩增快速检测鸡肉中的大肠杆菌O157:H

以大肠杆菌O157:H7的VT2基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立鸡肉中大肠杆菌O157:H7实时荧光定量环介导等温扩增(Rti-LAMP)检测方法。纯培养大肠杆菌O157:H7检测灵敏度达3.5 CFU/反应,需时45 min。模拟大肠杆菌O157:H7污染鸡肉样品,经37℃增菌4 h,用Whirl-pak无菌袋过滤离心得沉淀,提取样品DNA模板用于Rti-LAMP反应,检测大肠杆菌O157:H7灵敏度达140 CFU/g,整个检测流程约7 h。采用蒙脱石封闭的活性炭前处理污染鸡肉样品,不经增菌过程,结果表明:该Rti-LAMP检测方法灵敏度达12 CFU/g,整个检测耗时约4.5 h。市购鸡肉样品51份,以大肠杆菌国标检测法为对照,研究基于Rti-LAMP联合增菌或封闭活性炭预处理而不经增菌的两种方法,对比检测临床鸡肉样品大肠杆菌O157:H7污染率。结果:国标法和增菌的Rti-LAMP两种方法均检测到同一鸡肉样品为大肠杆菌O157:H7阳性,经封闭活性炭预处理而未经增菌的Rti-LAMP则检测到8份鸡肉样品大肠杆菌O157:H7阳性。研究表明,封闭活性炭预处理的Rti-LAMP检测方法较国标法更灵敏、快速、简便、特异地检测鸡肉中大肠杆菌O157:H7污染。     

大肠杆菌O157:H7特异基因的实时荧光定量PCR检测

为建立快速、特异的检测大肠杆菌O157:H7的实时荧光定量聚合酶链式反应(real time polymerase chainreaction,RT-PCR)方法,针对大肠杆菌O157:H7的特异基因rfbE设计一对特异引物,建立SYBR GreenⅠ实时定量PCR检测方法,并进行灵敏度、重复性和特异性实验,同时与常规PCR方法进行比较。结果显示所建立的SYBRGreenⅠ实时定量PCR方法可以快速、特异地检测出大肠杆菌O157:H7,细菌纯培养物中其灵敏度可达2×101CFU/mL,临床模拟污染肉样中能最低能检测到1×102CFU/mL的大肠杆菌O157:H7。与常规PCR方法相比,SYBR GreenⅠ实时定量PCR方法对临床样品中大肠杆菌O157:H7的检出率大大提高。本研究建立的SYBR GreenⅠ荧光定量PCR技术能快速准确、特异、敏感地检测大肠杆菌O157:H7。     

应用DPO技术检测食源性致病菌的多重PCR的研究

DPO是经特殊设计的特异性引物,本研究根据单增李斯特菌的prfA gene、O157的rfb Egene和副溶血弧菌的tlgene和志贺氏菌的ipaH gene设计四对DPO引物,采用多重PCR的方法,建立了检测单增李斯特菌、大肠杆菌O157:H7、副溶血弧菌和志贺氏菌的快速检测体系。实验结果表明:该体系特异性强,操作简单,为食源性致病菌的检测提供了新型的有效手段。     

三种食源性致病菌多重PCR检测方法的建立

目的:建立同时快速检测沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7的多重PCR方法。方法:根据沙门氏菌的invA基因、志贺氏菌的ipaH基因及肠出血性大肠杆菌O157∶H7的uidA基因设计3对引物,通过单因素实验、L9(34)正交实验优化反应体系,并对多重PCR扩增的敏感性进行分析。结果:3对引物能特异性扩增出495、620、252bp的目的片段;在最优多重PCR反应体系下,多重PCR检测3种致病菌的灵敏度达104CFU/mL;将该法应用于人工污染实验,可在5h内得到准确、稳定的检测结果。结论:该方法操作简单、检测特异性和灵敏度较高,能够实现对沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7 3种食源性致病菌的快速监控和诊断。     

多重PCR快速检测两种致泻性大肠杆菌方法的建立

本研究以肠出血性大肠杆菌O157∶H7及肠侵袭性大肠杆菌为目标菌,针对大肠杆菌共同基因uid A、肠出血性大肠杆菌O157∶H7的特异基因O-antigen、肠侵袭性大肠杆菌的特异基因ipa H设计3对引物,建立并优化了检测两种菌的多重PCR检测方法,进行了特异性验证和灵敏度分析,并应用于人工接种新鲜莴苣的检测中。实验结果表明,本研究建立的三重PCR快速检测两种致病菌的检出限为6.3×103CFU/m L,具有较好的灵敏度和特异性。利用所建立的多重PCR方法对人工接种肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌的新鲜莴苣进行检测,检出限为7.8×104CFU/m L。此方法能够对肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌两种食源性致病菌进行快速检测。     

IMSA技术快速检测肠出血大肠杆菌O157:H7方法的建立及应用

为了建立和评价一种适合食药监及基层检验部门使用的肠出血性大肠杆菌O157:H7快速检测方法,针对肠出血性大肠杆菌O157:H7特异性rfbE 基因,应用等温多自配引发扩增技术（Isothermal Multiple Self-matching-initiated Amplification,IMSA）,设计引物进行检测。对建立的方法进行特异性试验和灵敏度试验,与荧光定量PCR法进行比对;利用建立的IMSA法确定人工污染样本被检出的最短增菌时间和最低检测灵敏度;使用IMSA法和国标法对24份不同肉制品进行检验,评估两者的一致性。结果表明:该方法仅对肠出血性大肠杆菌O157:H7特异性扩增;IMSA法的灵敏度比qPCR法高,达8.9×102 CFU/mL;人工污染菌种样本最短增菌10 h可以被建立的IMSA法检出,检出限为9.1 CFU/25 g;24份肉制品样本的IMSA法与常规培养法结果一致性为100%。结论: IMSA技术检测肠出血性大肠杆菌O157:H7具有特异性强、灵敏度高、结果快速准确的特点,可在11 h内完成食品中O157:H7的检出,适用于食药监及基层检验部门进行肠出血性大肠杆菌O157:H7的快速检测。     

大肠杆菌O157:H7快速增菌培养基检测效果的研究

为了验证对于大肠杆菌O157:H7菌株特异性、复苏效果以及快速生长的特点,比较了3种培养基对于大肠杆菌O157:H7的增菌效果。研究结果表明,8h培养时间内,MicroFast~?快速增菌培养基对于冷损伤和热损伤的大肠杆菌O157:H7菌株具有良好的修复和增殖效果。MicroFast~?快速增菌培养基营养组分适合O157菌株的生长需求,在MicroFast~?快速增菌培养基中,大肠杆菌O157:H7菌株的倍增时间为13.5min,远高于m EC+n的28min,且MicroFast~?快速增菌培养基能够完全抑制部分G+细菌以及部分G-细菌的干扰,对于大肠杆菌O157:H7具有特异性的选择。故MicroFast~?快速增菌培养基对于大肠杆菌O157:H7菌株具有良好的选择特异性,能够短时间内(8h内)有效富集培养大肠杆菌O157:H7菌株,是一种能够实现大肠杆菌O157:H7菌株快速高效、特异增殖的培养基,将其结合检测金标卡,能够实现8h内快速检测,结果可信可靠。     

食品中大肠杆菌O157:H7的两种检测方法研究

研究食品中大肠杆菌O157∶H7的两种检测方法,PCR法引物针对特异性粘附毒素基因(eaeA)扩增检测,双抗夹心ELISA采用鸡抗O157∶H7特异性脂多糖(LPS)抗体(IgY)检测,结果显示两种方法对纯培养物的检测灵敏度都在103～104cfu/mL之间,特异性能满足食品检测需求;PCR法的敏感度较ELISA法高,且较省时。经以牛奶为参考的食品模拟样品(37℃增菌14h)验证两种检测方法的最低检出限均为2.5cfu/25mL样品。     

双抗体夹心ELISA法检测食品中出血性大肠杆菌O15

以灭活重组基因工程菌EHEC O157Δler/stx(p BR322::stx1/2B::eae)免疫新西兰大耳白兔获得的兔多抗作为捕获抗体,以针对出血性大肠杆菌O157主要毒力因子的特异性单抗2H9为检测抗体,建立双抗体夹心ELISA方法并绘制标准曲线用于食品中出血性大肠杆菌O157的定量分析。标准曲线线性方程为y=0.3479x-0.4121,R~2=0.9862。该ELISA的重复性变异系数小于5%,稳定性良好。应用该方法测得出血性大肠杆菌O157∶H7 EDL933的最小检出限为10~3cfu/m L。食品样品接种实验表明:牛肉或猪肉样品接种量为0.08 cfu/g,增菌8 h后可以检出阳性反应;蔬菜和牛奶样品接种量为0.12 cfu/g(m L),增菌10 h后可以检出阳性反应。     

纺织品中大肠杆菌O157:H7的检测方法

为建立一种灵敏、特异、快速、高效地鉴定纺织品基质中大肠杆菌O157:H7的方法,筛选出大肠杆菌O157:H7特有的rfbE基因保守序列,设计PCR特异性引物和荧光双标记探针,结合免疫磁珠技术,集成创新开发一种目标菌低浓度、不可培养情况下的样品中高效、快速富集分离大肠杆菌O157:H7的技术,建立了一种针对纺织品基质的免疫磁珠富集-实时荧光定量PCR方法,其检测下限可达8 CFU/mL。利用该方法对收集到的30份阳性样品进行鉴定,鉴定结果与传统方法结果100%吻合。结果表明,新建方法稳定性强,实现了纺织品中大肠杆菌O157:H7的快速灵敏鉴定。     

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。     

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.     

PCR-免疫胶体金试纸条方法检测食品中 肠出血性大肠杆菌O157:H7

目的建立PCR-免疫胶体金试纸条法快速检测食品中肠出血性大肠杆菌O157:H7的分析方法。方法通过设计特异性引物建立肠出血性大肠杆菌O157:H7 PCR检测方法并使用免疫胶体金技术以及双抗体夹心法建立PCR产物快速检测试纸条并设计核酸检测展开液;将1株大肠杆菌O157:H7标准菌株和7株其他常见食源性致病菌作为试验菌株,用试验菌株检测PCR-免疫胶体金试纸条方法的检测特异度,并比较PCR-免疫胶体金试纸条法和PCR-琼脂糖凝胶电泳法的检测敏感度。结果 PCR-免疫胶体金法具有良好的特异度,灵敏度比标准琼脂糖凝胶电泳法高100倍。结论本文建立的肠出血性大肠杆菌O157:H7检测PCR-免疫胶体金试纸条法特异度好,灵敏度高,价格低廉,适用于食品中肠出血性大肠杆菌O157:H7的检测。     

多重PCR检测冷却肉中的3种致病菌

分别针对沙门氏菌(Salmonella choleraesuissubs.choleraesuis,S)编码DNA结合蛋白的基因hns、大肠O157:H7(Escherichia coliO157:H7,E)编码外膜蛋白紧密素的基因eaeA和单核细胞增生李斯特氏菌(Listeria monocytogenes,L)溶血素O基因Hly设计3对引物,建立同步检测肉制品中3中致病菌的多重PCR方法。通过对多重PCR特异性和灵敏度进行分析,对多重PCR反应条件进行优化,结果表明,此方法简便、快速,可使混菌检测灵敏度达到103cfu/mL。     

基于内参的大肠埃希氏菌O157∶H7实时荧光定量PCR快速检测方法的建立

针对大肠埃希氏菌O157∶H7 rfbE和Flic靶基因保守序列,设计特异性引物和探针,优化反应体系,并加入内参（internal amplification control,IAC）,建立能够实时监控反应过程的荧光定量聚合酶链式反应（polymeasechain reaction,PCR）检测方法。结果表明,该方法对E. coli O157∶H7基因组DNA的最低检测限为1 pg/μL;对含有靶基因质粒的最低检测限为103 copies/μL;对细菌的最低检测限为5×103 CFM/mL;Ct值与模板拷贝数均呈良好的线性关系（R2=0.999）。人工污染实验结果表明,在初始菌量为7 CFM/25 g时,采用水洗加试剂盒法提取DNA,E. coli O157∶H7在增菌6 h后即可检出;而采用水煮法提取DNA,则在增菌10 h后方可检出。建立的E. coli O157∶H7实时荧光定量PCR方法,既能有效检测食品中O157∶H7,又能实时监测PCR反应过程,有效防止“假阴性”的发生。     

Detection, occurrence, and characterization of Escherichia coli O157:H7 from raw ewe's milk in Spain

A study was carried out in the Castilla y León region of Spain to investigate the presence of Escherichia coli O157:H7 in raw ewe's milk samples collected from several cheese factories during 1 year. All specimens were examined for E. coli O157:H7 by selective enrichment at 41.5 +/- 1.0 degrees C, after both 6 and 22 h of incubation, and then immunomagnetically separated and plated on cefixime-potassium tellurite-sorbitol MacConkey agar. No growth was obtained in the enrichment broth after a 6-h incubation. Presumptive colonies obtained after 22 h of incubation were screened by a multiplex PCR assay for the presence of rfbO157 and fliCH7 genes. Of all the ewe's milk samples studied, three were positive for E. coli O157:H7. The E. coli O157:H7 strains that were positive for the rfbO157 and fliCH7 genes were then analyzed by multiplex PCR for the presence of virulence genes (stx1, stx2, ehxA, and eaeA). All E. coli O157:H7 isolates were Shiga toxigenic and harbored additional genes related to virulence (ehxA and eaeA). The predominant Stx toxin type was stx2. These results demonstrate that raw ewe's milk used in cheesemaking may be sporadically contaminated with E. coli O157:H7 strains that are potentially pathogenic for humans.     

大肠埃希O157:H7等致病菌多重PCR法快速检测试剂盒的研制

目的:研制一种能够同时快速检测大肠埃希O157:H7、志贺氏菌和致病性蜡样芽孢杆菌的三重PCR试剂盒.方法:以大肠埃希O157:H7的hlyAB基因、痢疾志贺氏菌的IpaH基因和致病性蜡样芽孢杆菌的hblA基因作为目的基因片段O157:H7、痢疾志贺氏菌和致病性蜡样芽孢杆菌的特异性抗原基因序列,分别设计1对引物,并对其反应条件进行优化,建立1种多重PCR检测试剂盒.结果:本试剂盒可准确检测出生肉和即食肉制品中的上述3种致病菌,O157:H7检出极限为19.8 cfu/mL,志贺氏菌检出极限为17 cfu/mL,蜡样芽孢杆菌检出极限为17.7 cfu/mL.可在5 h内完成全部反应过程,得出检测结果.结论:本试剂盒在理论和实际应用方面均具有优越性,能够同时检测上述3种病原菌,可用于食品及其原料的生物安全检测,也可用于兽医临床诊断.     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.