中国部分食品中肠炎沙门菌分离株的PFGE分子型别分析研究

目的 研究中国食品中肠炎沙门菌分离株的分子谱别.方法 用脉冲场凝胶电泳分型技术对从中国11个省市食品中分离的51株肠炎沙门菌进行分子分型.结果 肠炎沙门菌经2种内切酶Xba I、Spe I处理后,均分为11个不同的带型.两种内切酶的分型结果不相同,但其中有部分交叉.2种内切酶的PFGE分型都有2个主要的带型,分别涵盖了分离株的70%,并在两型之间有部分兼容性.通过双酶系统的双重分型,还可以将其中的型别进一步区分为亚型.在各地区之间菌株的型别分布无明显规律,在各种食品之间菌株的型别分布,SpeI酶切后的结果更有规律.分离自羊肉中的4株菌经XbaI、Spe I酶切表现为同-PFGE型别,在Xba I分型结果中为XF型,在Spe I分型结果中为SE型,揭示了一定的亲缘关系.结论 脉冲场凝胶电泳分型技术是肠炎沙门菌分子分型的有效手段.     

一起食物中毒病原菌斯坦利沙门菌的分子分型及耐药性分析

目的对引起跨区域食物中毒的斯坦利沙门菌分离株进行耐药性检测和分子分型分析,追溯并明确病因食品,为该菌防控工作提供科学依据。方法采用微量肉汤稀释法对15株斯坦利沙门菌进行18种抗生素敏感性试验,同时对菌株基因组DNA经限制性内切酶Xba I酶切后进行脉冲场凝胶电泳(PFGE)分子分型,并与数据库中其他沙门菌比较。结果此次疫情分离菌株中食品分离株和腹泻患者分离株血清型一致,耐药谱基本一致。经PFGE溯源分析后具有100%同源的PFGE带型,数据库中无相同带型的菌株。结论本起跨区域食物中毒是由斯坦利沙门菌污染的煮花生引起,分离菌株对红霉素耐药,对氯霉素处于中介,对其他抗生素敏感。     

脉冲场电泳在两起同期异地肠炎沙门菌食物中毒分子流行病学调查中的应用研究

目的 探讨两起相距210多公里乡镇同期发生的肠炎沙门菌食物中毒分离株之间的分子流行病学关系.方法 将两起食物中毒事件患者和剩余食物分离到的8株肠炎沙门菌进一步鉴定培养,挑取单个菌落增菌,用限制性内切酶Xba I消化酶切后进行脉冲场凝胶电泳,获得的分子分型指纹图谱,运用Bionumerics 5.1进行聚类分析.结果 指纹图谱聚类显示8株肠炎沙门菌分为两个分子型,D乡食物中毒分离到的5株肠炎沙门菌有相同的指纹图谱;P镇的3株分离株图谱也一致;两起食物中毒分离株图谱之间有4个条带的差异.结论两起食物中毒的暴发虽然都是肠炎沙门菌引起的,但具有不同的分子型别,食物中毒不是由同一种污染的食物源引起;脉冲场凝胶电泳可有效应用于食物中毒溯源分析及肠炎沙门菌分子流行病学研究.     

椰毒假单胞菌酵米面亚种脉冲场凝胶电泳分型的研究

目的建立椰毒假单胞菌酵米面亚种的脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)分型方法,并对29株分离株进行PFGE分型研究。方法分别选择5种限制性内切酶(XbaⅠ、SpeⅠ、NotⅠ、SmaⅠ、AscⅠ)酶切椰毒假单胞菌酵米面亚种染色体DNA以进行PFGE分析,并利用BioNumerics软件对分离株的指纹图谱进行聚类分析。结果NotⅠ、SmaⅠ和AscⅠ的酶切片段过小,而XbaⅠ酶切片段的大小适中但数量过多,不能获得清晰可辨的图谱,均不适于椰毒假单胞菌酵米面亚种的PFGE分型。SpeⅠ酶切的PFGE条带数量和大小适中、指纹图谱清晰可辨,对椰毒假单胞菌酵米面亚种具有足够的分辨率。结论本研究建立的PFGE分型方法可应用于椰毒假单胞菌酵米面亚种的分子分型和溯源。     

浙江省369份淡、海水鱼香港海鸥型菌污染状况研究

目的 掌握浙江省淡、海水鱼中香港海鸥型菌污染状况，为防止食源性香港海鸥型菌痛的发生和流行提供科学依据。方法 用改良头孢哌酮麦康凯琼脂分离，API20NE鉴定香港海鸥型菌。K-B纸片扩散法作耐药性测定。通用引物扩增16S rDNA并与香港海鸥型菌HKU1株开展同源性比较。结果 369份样品检出香港海鸥型菌18株。阳性率为4．88％，其中，鲤鱼检出率为25．00％，草鱼的阳性率为10．26％，海水鱼以及鲫鱼、鲢鱼、扁鱼等淡水鱼中未分离出。分离的香港海鸥型菌16SrDNA序列与HKU1株仅相差1～2个碱基，同源性在99．6％～100．0％之间；18株菌对头孢拉定、万古霉素、甲硝唑、克林霉素、头孢哌酮完全耐药，对红霉素、亚胺培南、克拉霉素、氨曲南、庆大霉素、丁胺卡那霉素、三甲氧苄氨嘧啶、四环素、氯霉素、多粘菌素B、环丙沙星完全敏感，对头孢噻吩、胺苄西林、头孢曲松的耐药性分别为83．33％、61．11％和5，56％。结论浙江省存在香港海鸥型菌污染源，建议相关监督部门应加大这类产品的管理力度。防止香港海鸥型菌病在我省的发生和传播。     

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。     

PFGE与16S rDNA对阪崎克罗诺杆菌分型的研究

研究脉冲场凝胶电泳(PFGE)与16S rDNA序列分析在阪崎克罗诺杆菌分型中的应用。对分离自不同来源的10株阪崎克罗诺杆菌进行16S rDNA序列扩增及测序,并构建系统发育树进行聚类分析。再利用限制性内切酶Xba I对这10株菌酶切后,进行PFGE电泳分型。16S rDNA序列分析显示,所有分离菌株与ATCC标准菌株在同一系统发育分支下,其序列相似性达到98.37%,并且不能进一步分型。而PFGE分型结果显示,10株分离菌株和2株标准菌株共分成11种PFGE基因型,说明PFGE的分型能力明显优于16S rDNA序列分析,部分菌株的基因型与分离来源具有一定的相关性,所以PFGE分型方法可用于阪崎克罗诺杆菌分子分型及溯源的研究。     

进出境食品中单核增生李斯特氏菌的血清分型与脉冲场凝胶电泳分型分析研究

目的 研究进出境食品中的单核增生李斯特氏菌（LMO）的血清分型与脉冲场凝胶电泳分型的特性及其关系。方法 采用标准方法对39株分离的LMO进行PFGE分型,分别用ApaI和Asc I酶切,电泳图谱进行聚类分析,以上菌株同时进行血清分型检测。结果 39株LMO分成6个血清型;经ApaI酶切后,分成29种带型,相似度在57.4%~100%;Asc I酶切后,分成34种带型,相似度在48.6%~100%。讨论 AscI酶切的PFGE分离效果优于ApaI酶切,与血清分型的一致性低于ApaI酶切;PFGE分型效果优于血清分型。     

进出境食品中单核增生李斯特菌的血清分型与脉冲场凝胶电泳分型分析

目的研究进出境食品中的单核增生李斯特菌(LMO)的血清分型与脉冲场凝胶电泳(PFGE)分型的特性及其关系。方法采用标准方法对39株分离的LMO进行PFGE分型,分别用ApaI和Asc I酶切,电泳图谱进行聚类分析,以上菌株同时进行血清分型检测。结果 39株LMO分成6个血清型;经ApaI酶切后,分成29种带型,相似度在57.4%～100%;Asc I酶切后,分成34种带型,相似度在48.6%～100%。讨论 AscI酶切的PFGE分离效果优于ApaI酶切,与血清分型的一致性低于ApaI酶切;PFGE分型效果优于血清分型。     

香港海鸥型菌(Laribacter hongkongensis)的鉴定方法研究现状

香港海鸥型菌是从香港肝硬化病人体内分离得到的新细菌菌属，被认为是致腹泻病原菌。为给相关的研究提供参考，介绍香港海鸥型菌选择性培养基的筛选，生物学特征检测方法，基因序列分析检测方法中的16s rRNA寡核苷酸序列分析定性鉴定核糖分型(ribotyping)与脉冲电场电泳，自动化细菌鉴定系统。     

腹泻患儿粪便中沙门菌的血清型、药敏分析及脉冲场凝胶电泳分子分型研究

目的 研究2019年北京市朝阳区0-10岁腹泻患儿粪便中分离出的沙门菌的血清型、PFGE分子分型研究及耐药特点。方法 对分离自腹泻患儿病例的47株沙门菌进行血清分型,采用微量肉汤稀释法进行27种抗生素的药敏实验;采用PFGE脉冲场凝胶电泳进行指纹图谱分型研究。结果 47株沙门分为9种血清型,优势血清型两种,分别为肠炎沙门菌23株占48.94%,鼠伤寒沙门菌14株占29.79%。47株沙门菌对磺胺异恶唑耐药率(57.45%)最高,其次为氨苄西林(48.94%)和链霉素(48.94%)。23株肠炎沙门菌可分成8个PFGE指纹图谱,15株鼠伤寒沙门菌可分成14个PFGE指纹图谱。结论 北京市朝阳区0-10岁儿童食源性沙门菌血清主要为肠炎沙门菌和鼠伤寒沙门菌,各血清型的耐药性有所不同,PFGE指纹图谱呈多样性。     

Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.     

云南省食源性金黄色葡萄球菌脉冲场凝胶电泳分型分析

目的采用脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法分析云南省2013～2015年食源性金黄色葡萄球菌分子分型带型,初步建立金黄色葡萄球菌PFGE分型的数据库。方法用限制性内切酶Smal I酶切113株金黄色葡萄球菌染色体DNA以进行PFGE分析,并利用BioNumerics软件对分离株的指纹图谱进行聚类分析。结果 113株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在56.67%～100%之间,经聚类分析得到89个PFGE型别。结论建立云南省食源性金黄色葡萄球菌PFGE分型数据库,PFGE带型呈现多样性,对今后云南省金黄色葡萄球菌引起食物中毒的诊断和溯源工作有重要意义。     

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

利用ISSR分析贵州部分烟草品种(系)的遗传关系

利用ISSR标记,对12个贵州地方品种(系)和5个贵州主要种植品种分析亲缘关系。结果显示,有23条ISSR引物在烟草上能扩出清晰条带,其中9条引物能扩增出明显多态,能把17个品种(系)区分开。利用NTSYS 2.0软件对多态条带进行统计;用SimQual计算遗传相似系数,遗传相似系数在0.2127~0.7872之间变动。根据UPGMA进行聚类分析结果表明,17个品种(系)可以分为3个组群。     

单核细胞增生李斯特菌的毒力基因检测及PFGE分型的研究

目的了解福建省食品中单核细胞增生李斯特菌携带hly、plcA、plcB和prfA毒力基因的情况及脉冲场凝胶电泳(PFGE)的分型情况。方法将hly、plcA、plcB和prfA基因作为靶序列选取4对引物,通过聚合酶链反应(PCR)检测61株单核细胞增生李斯特菌和2株可疑单核细胞增生李斯特菌的毒力基因,用PulseNet单核细胞增生李斯特菌标准方法进行7株单核细胞增生李斯特菌的PFGE分子分型。结果61株单核细胞增生李斯特菌毒力基因为hly 、plcA 、plcB 和prfA ,2株可疑单核细胞增生李斯特菌的毒力基因分别为hly-、plcA 、plcB-、prfA-和hly-、plcA-、plcB-、prfA-。7株单核细胞增生李斯特菌的PFGE分为5个型。结论实验结果表明福建省食品中分离到的单核细胞增生李斯特菌均含有hly、plcA、plcB和prfA基因,属于致病株。对2株可疑单核细胞增生李斯特菌进行了进一步的鉴定,排除了单核细胞增生李斯特菌,该毒力基因检测方法可用于可疑单核细胞增生李斯特菌的进一步鉴别。7株菌中有两对2株PFGE型别一致,一致的菌株来自不同年份不同销售地点的同一品牌,应用PFGE方法可以进一步调查该厂冻鸡肉中单核细胞增生李斯特菌的传播途径和污染源。     

苏州市食源性金黄色葡萄球菌耐药分析及基因分型

目的 对苏州市2014~2017年分离的食源性金黄色葡萄球菌进行耐药谱和分子分型分析, 初步建立PFGE(pulsed-field gel electrophoresis)分型数据库。方法 采用微量肉汤稀释法检测金黄色葡萄球菌(Staphylococcus aureus, SAU)对19种常用的抗生素的耐药性, 采用Sma I进行酶切, 脉冲场凝胶电泳进行分子分型。菌株指纹图谱用BioNumerics v6.6软件进行分析。结果 81株SAU中检出10株耐甲氧西林金黄色葡萄球菌(methicillin-resistance Staphylococcus aureus, MRSA), 检出率12.3%。有72株菌存在不同程度的耐药, 耐药率达到88.9%, 其中对青霉素的耐药率最高(82.7%), 其次是氨苄西林(66.7%), 其后是克林霉素和红霉素(均为50.6%)。耐受3种和3种以上药物的多重耐药菌株共38株, 多重耐药率达46.9%。MRSA的多重耐药率高于甲氧西林敏感型金黄色葡萄球菌(methicillin-sensitive Staphylococcus aureus, MSSA), 差异具有统计学意义(P<0.05)。除8株菌不可被PFGE分型外, 其余73株菌可以被分为37个不同型别, 其中有4个优势型别共27株菌, 在可分型菌株中占比达37.0%。这些同源菌株分离自不同时间和地点, 有些还存在一定程度的变异。结论 食源性金黄色葡萄球菌耐药现象普遍存在, 多重耐药率较高, PFGE型别较为多样但有优势型别存在, 耐药谱和PFGE型别关联性较差。     

北京市顺义区2起产气荚膜梭菌食物中毒病原学分析

目的研究2起食物中毒事件中分离到的产气荚膜梭菌(Clostridium perfringens,CP)的病原学特征。方法采集2起食物中毒事件涉及的病例、厨师、烹饪用具和可疑食品等标本或样品,并对其进行常见病毒和细菌的实时荧光定量聚合酶链式反应(real-time PCR)初筛及细菌分离培养。计数标本或样品中CP数量,并对分离到的CP菌株进行毒力基因、脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)和耐药性检测。结果经过real-time PCR和细菌分离培养,在部分病例标本以及食品和涂抹样品中检出CP,其他常见病毒和细菌均为阴性。大部分病例粪便标本的CP计数结果大于1.0×10~6 CFU/g。10株CP分离菌株均携带毒力基因;菌株PFGE分为7个带型,其中第一起事件有3株为同一带型,第二起有2株为同一带型;菌株对亚胺培南、甲硝唑、头孢曲松和青霉素敏感,6株对克林霉素耐药、1株中介,6株对氯霉素中介。结论 PCR可应用于CP毒力基因的快速筛查,PFGE也可辅助判定CP引起的食物中毒事件,同时应加强对CP的日常监测和其耐药性的防范。     

Persistent and nonpersistent Listeria monocytogenes contamination in meat and poultry processing plants

Contamination analysis of persistent and nonpersistent Listeria monocytogenes strains in three meat processing plants and one poultry processing plant were performed in order to identify factors predisposing to or sustaining persistent plant contamination. A total of 596 L. monocytogenes isolates were divided into 47 pulsed-field gel electrophoresis (PFGE) types by combining the restriction enzyme patterns of AscI (42 patterns) and ApaI (38 patterns). Persistent and nonpersistent strains were found in all plants. Nonpersistent PFGE types were found mostly at one sampling site, with the processing environment being the most common location, whereas the persistent strains were found at several sampling sites in most cases. The processing machines were frequently contaminated with persistent L. monocytogenes PFGE types, and it was of concern that surfaces having direct contact with the products were contaminated. The role of the processing machines in sustaining contamination and in contaminating the products appeared to be important because the final product of several processing lines was contaminated with the same L. monocytogenes PFGE type as that found in the processing machine. The proportion of persistent PFGE types in heat-treated products was eight times higher than in the raw products, showing the importance of the persistent PFGE types as contaminants of the final heat-treated products. The contamination status of the processing lines and machines appeared to be influenced by the compartmentalization of the processing line, with poor compartmentalization increasing L. monocytogenes contamination. The separation of raw and post-heat treatment areas seemed especially important in the contamination status of post-heat treatment lines.