Fla-DGGE法对食品中空肠弯曲菌和结肠弯曲菌的检测和分型

本文应用鞭毛蛋白基因flaA和flaB的变性梯度凝胶电脉(denaturing gradient gel electrophoresis,DGGE)方法对食品中空肠弯曲菌和结肠弯曲菌进行检测和分型。采用RBB(rpeated bead beating)、CTAB和丙酮-氯仿抽提三种方法提取样品基因组后进行fla-DGGE检测,结果完全一致。10份生鲜鸡、鸭肉样品中有8份含有空肠弯曲菌或结肠弯曲菌,其中3份含有两种或两种以上的空肠弯曲菌或结肠弯曲菌或它们的不同型别。克隆测序后结果表明,有7份样品被同一种空肠弯曲菌所污染,其中3份样品还被同一种结肠弯曲菌所污染,1份样品被污染情况严重,检测到含有一种结肠弯曲菌和三种空肠弯曲菌。Fla-DGGE方法快速、准确、灵敏度高,可用于食品中空肠弯曲菌和结肠弯曲菌的快速检测和分型。     

食源性空肠弯曲菌的分子分型及其遗传多样性分析

为了解在2012~2014年从中国不同城市分离到的食源性空肠弯曲菌的遗传多样性特征,本研究采用多序列位点分型(multilocus sequencing typing,MLST)的方法对分离到的33株食源性空肠弯曲菌进行分子分型。研究中,对33株菌株的管家基因使用7对不同的引物进行PCR扩增,并对扩增的产物使用凝胶电泳鉴定后进行测序。将测序结果同Pub MLST中Campylobacter数据库进行比对,以获得对应菌株ST型,并提交新的ST型数据。利用其MLST数据构建进化树和最小生成树。结果显示33株食源性空肠弯曲菌可以分为27个ST型,其中有14种为新的ST型,可形成11种克隆复合体,优势克隆复合体为CC22和CC45,部分菌株的CC型也曾在临床上发现。共存在91种核苷酸多态性位点,部分等位基因之间存在重组。这表明在2012-2014年间分离得到的菌株具有丰富的遗传多样性,并且有潜在致病风险。     

空肠弯曲菌致病分子机制研究进展

空肠弯曲菌(Campylobacter jejuni)是人类的食源性病原菌,感染后主要引起急性肠炎,与人类格林-巴利综合症也有密切关系.研究表明,空肠弯曲菌致病性是多种毒力因子共同作用的结果.作者从分子生物学研究水平综述空肠弯曲菌黏附、定植、侵入、产细胞毒素、分子模拟等致病机制.     

空肠弯曲菌flhA突变株的构建及其功能研究

flhA是空肠弯曲菌鞭毛输出装置的关键基因,通过构建Campylobacter jejuni flhA突变株HXW001,进行毒力相关表型研究和分析。结果显示HXW001生长性能稳定,但其鞭毛和运动能力丧失,生物膜形成和自身凝集现象明显下降。RT-PCR实验证明flhA对鞭毛丝主要结构蛋白基因flaA的表达有一定的调控功能。研究表明flhA是空肠弯曲菌鞭毛生成和运动能力重要的分子基础,与空肠弯曲菌致病性密切有关。     

双重PCR 方法检测沙门氏菌和空肠弯曲菌

为建立能够同时检测食品中沙门氏菌和空肠弯曲菌的双重PCR 方法。采用沙门氏菌鞭毛基因fimY 和空肠弯曲菌马尿酸酶基因hipO 设计特异性引物,并对影响PCR 扩增的主要因素——引物浓度、退火温度、Mg2+ 浓度因素进行优化,比较单一PCR 和双重PCR 的检测效果。结果表明：采用单一PCR 法检测沙门氏菌和空肠弯曲菌时,灵敏度分别可达到3.98pg 和4.05pg;而采用双重PCR 检测时,灵敏度较单一PCR 法有所下降,沙门氏菌和空肠弯曲菌检出限量分别为398pg 和40.5pg。本研究建立的特异性强和灵敏度高的双重PCR 检测方法,可为实现食品中沙门氏菌和空肠弯曲菌的同时检测提供新方法。     

多重PCR鉴定动物源空肠弯曲菌和结肠弯曲菌方法的建立

建立鉴定空肠弯曲菌和结肠弯曲菌的多重PCR(mPCR)方法。方法 分别以16S rRNA、马尿酸酶和16S-23S rRNA基因为靶序列设计特异性引物,建立多重PCR方法检测37株菌株样品,同时采用ⁱⁿgene CAM nested PCR检测试剂盒检测验证,进行结果比较分析。结果 该多重PCR方法可扩增出空肠弯曲菌和结肠弯曲菌的特异性条带,其他对照菌株均未扩增出条带,具有较好的特异性;检测敏感性可达0.81pg/μl空肠弯曲菌DNA,0.93pg/μl结肠弯曲菌DNA。多重PCR方法和试剂盒检测结果的符合率为100%,二者与国标GB/T 4789.9—2008方法的符合率达97%以上。结论 本试验建立的多重PCR方法操作快速方便、节约试验成本,具有较好的特异性、敏感性和重复性,可用于弯曲菌的鉴定。     

空肠弯曲菌与食品

近几年来空肠弯曲菌(Campylahacterjej－uni)已被世界各国公认为是引起人类急性腹泻,特别是婴儿腹泻的重要病原菌。空肠弯曲菌可以多种方式从动物宿主传播给人,也可通过直接接触污染的动物宰体传播。但是,更多的是通过摄入污染的食物或水而间接传播的,...     

动物源弯曲菌分离纯化鉴定方法优化

【目的】弯曲菌是重要的食源性人畜共患病原菌,通常难以诊断,而动物源方向缺少系统的检测分析方法及相应的优化。为了获得更适宜分离动物来源的空肠弯曲菌和结肠弯曲菌,提高分离效率,降低分离成本,特开展方法优化,填补动物源弯曲菌分离方法建设的空白,为耐药菌株的有效追踪溯源和风险评估提供依据。【方法】对已有的空肠弯曲菌和结肠弯曲菌分离纯化鉴定方法Preston肉汤和Bolton肉汤增菌;CCDA选择性培养分离、弯曲菌显色培养分离、Skirrow选择性培养分离;生化鉴定和分子生物学鉴定做了筛选优化,针对目前存在的分离纯化和鉴定方法进行了调查比较,得到了更适合于健康动物来源的粪便和盲肠中的分离方法。【结果】牛血清预增菌、CCDA选择性分离、弯曲菌显色培养基纯化、哥伦比亚血琼脂进行扩增及分子学方法鉴定,同时建议采用厌氧条件进行培养。该流程简单易行,成本较低,适合于绝大多数实验环境条件,是获取弯曲菌的首选流程,便于推广实施。【结论】通过将现研究阶段存在的大多数检测方法进行了综合分析,整理出一套针对于动物来源的粪便及肠道内容物的检测分析方法,对健康动物中弯曲菌的分离效率能够提高30%,同时空肠弯曲菌和结肠弯曲菌的分离效率无明显差异。     

空肠弯曲菌在牛奶中的存活和对热敏感性的实验

空肠弯曲菌(Campylobacter jejuni)是引起人类急性腹泻和食物中毒的一种重要病原菌,已知牛奶可被其作为媒介而引起疾病的爆发。因此该菌在牛奶中的存活情况,以及巴氏消毒法是否能杀死该菌以保证这种奶的安全是重要的。为此,笔者进行了这方面的实验研究,现报告如下。     

PCR技术检测空肠弯曲菌的研究进展

空肠弯曲菌(Campylobacter jejuni)是一种人畜共患病病原菌,可以使人和动物引发多种疾病。目前,检测C.jejuni采用的国标方法是传统的培养法,但C.jejuni培养条件苛刻,且培养法存在操作繁琐、特异性不强、费时等缺点。聚合酶链式反应(polymerase chain reaction,PCR)以其快速、准确、灵敏度高、特异性强的特点,现已广泛应用于C.jejuni的检测,并成为目前快速检测临床与食品中C.jejuni最常用的的方法。本文综述了近年来利用RCR技术,包括常规PCR、多重PCR、巢式PCR、实时荧光定量PCR、PCR-酶联免疫吸附法、最大几率数-PCR、PCR-限制性片段长度多态性、PCR-变性高效液相色谱、PCR-变性梯度凝胶电泳和磁捕获-荧光PCR方法检测C.jejuni的研究进展,并针对这些PCR技术的原理、检测效果、优点和缺点等方面进行了分析比较,为有效控制和预防该菌引起的疾病提供重要信息。     

Bacterial dormancy in Campylobacter: abstract theory or cause for concern?

For the past 100 years, since the birth of modern microbiology, this discipline has predominantly relied on the ability to culture micro-organisms in vitro on artificial synthetic culture media under controlled conditions in the laboratory. However, sometimes it is not possible to detect foodborne pathogens using such conventional techniques. Employment of these techniques can also lead to a delay in detection of pathogens. The 'viable but non-culturable' (VNC) cellular form has been demonstrated in Campylobacter jejuni , representing a resting or dormant stage, which is induced through cell stress including starvation. This form is extremely difficult to detect and generally requires complex and sophisticated technology which is usually not available in most routine food microbiology laboratories. This review aims at examining the role of this cell form in Campylobacter , including their historical evolution, formation, physiology, detection and to discuss the challenges that this form presents to food safety.     

Campylobacter jejuni: A Review of its Characteristics,Pathogenicity, Ecology,Distribution, Subspecies Characterization and Molecular Methods of Detection

Campylobacter jejuni is considered to be the leading cause of enteric illness in the United States and other industrialized nations, causing mild to severe symptoms including serious infections of the extremities and permanent neurological symptoms. The organism is a cytochrome oxidase positive, microaerophilic, curved Gram-negative rod exhibiting corkscrew motility and is carried in the intestine of many wild and domestic animals, particularly avian species including poultry, where the intestine is colonized resulting in healthy animals as carriers. Wildlife have long been considered an infectious reservoir for campylobacters because of their close association with and contamination of surface waters. This review deals with the areas of: phenotypic characteristics of C. jejuni and related human pathogenic species of Campylobacter, their ecological distribution, virulence factors, isolation of C. jejuni from foods, serotyping of Campylobacter isolates, bacteriophage typing, molecular methods of detecting and typing campylobacters, the viable but nonculturable state of campylobacters, the coccoid form of C. jejuni and immunomagnetic capture of C. jejuni.     

Phenotypic Characters and Molecular Epidemiology of Campylobacter Jejuni in East China

In this study, we investigated the distribution, phenotypic and molecular typing characters of Campylobacter jejuni in domestic fowl, and livestock populations in East China, to provide some reference for researches on its molecular epidemiology. A total of 1250 samples were collected from different animal sources, and C. jejuni strains were then isolated and tested for antibiotic sensitivity. Antibiotics‐resistance gene and pathogenic genes were detected by polymerase chain reaction. Phylogenic analysis on the C. jejuni strains was performed by multilocus sequence typing (MLST) method. The results showed that 108 out of the 1250 samples (mean 8.64%) were C. jejuni positive. These 108 C. jejuni strains were highly sensitive to antibiotics such as chloramphenicol, amoxicillin, amikacin, cefotaxime, and azithromycin, whereas they were highly resistant to antibiotics such as cefoperazone, cotrimoxazole, cefamandole, sulfamethoxazole, and cefradine. Pathogenicity related gene identification indicated that the mean carrying rate of adhesion related gene cadF and racR, flagellin gene flaA, toxin regulating gene cdtA, cdtB, cdtC, wlaN and virB11, heat shock proteins and transferring proteins related genes dnaJ and ceuE, CiaB and pldA were 92.45%, 38.69%, 73.58%, 71.70%, 52.83%, 96.23%, 12.26%, 1.89%, 0.94%, 65.09%, 39.62% and 9.43%, respectively. A total of 58.82% of these strains contained more than 6 pathogenicity‐related genes. MLST typed 58 ST types from the 108 isolated C. jejuni strains, including 24 new types, and ST‐21 was the major type, accounting for 39.3% of the total strains.     

Campylobacter jejuni: A Review of its Characteristics, Pathogenicity, Ecology, Distribution, Subspecies Characterization and Molecular Methods of Detection

Campylobacter jejuni is considered to be the leading cause of enteric illness in the United States and other industrialized nations, causing mild to severe symptoms including serious infections of the extremities and permanent neurological symptoms. The organism is a cytochrome oxidase positive, microaerophilic, curved Gram-negative rod exhibiting corkscrew motility and is carried in the intestine of many wild and domestic animals, particularly avian species including poultry, where the intestine is colonized resulting in healthy animals as carriers. Wildlife have long been considered an infectious reservoir for campylobacters because of their close association with and contamination of surface waters. This review deals with the areas of: phenotypic characteristics of C. jejuni and related human pathogenic species of Campylobacter, their ecological distribution, virulence factors, isolation of C. jejuni from foods, serotyping of Campylobacter isolates, bacteriophage typing, molecular methods of detecting and typing campylobacters, the viable but nonculturable state of campylobacters, the coccoid form of C. jejuni and immunomagnetic capture of C. jejuni.     

动物性食品源空肠弯曲杆菌二重PCR检测方法的建立及应用

根据GenBank空肠弯曲杆菌(Campylobacter jejuni,Cj)的16S rDNA及hipO(编码马尿酸酶基因)序列设计两对特异引物,建立检测动物性食品源Cj的二重PCR方法,并应用于样品检测。结果显示只对Cj能特异的扩增出699bp和366bp两个基因片段,而大肠杆菌、沙门氏菌等其他11种细菌均未扩增出条带;Cj标准株ATCC33560的16S rDNA及hipO序列与GenBank其他Cj的相应序列具高度相似性(分别为99.7%~99.9%,98.1%~99.7%);该方法可在27h内完成,其灵敏度为2.4~16 CFU/mL;四川省雅安市鸡肉、猪肉、牛肉和牛奶样品中的Cj阳性率分别为38.0%(19/50)、28.3%(15/53)、17.1%(6/35)和8.6%(4/46)。     

一种检测活的非可培养状态空肠弯曲菌的新方法

为提高活的非可培养状态空肠弯曲菌的检出率,建立了一种免疫磁珠捕获-荧光聚合酶链式反应法。该法包括应用免疫磁珠技术制备弯曲菌免疫磁珠,以直接捕获受检样品中的空肠弯曲菌,并设计了检测空肠弯曲菌马尿酸酶(hipO)基因的引物与荧光标记探针。结果表明:磁捕获-荧光聚合酶链式反应法可直接检测空肠弯曲菌,无需增菌培养,具有灵敏度高、选择性好、简便、快速等特点,可在24h内完成样品中活的非可培养状态空肠弯曲菌的检测。     

Comparison between conventional and qPCR methods for enumerating Campylobacter jejuni in a poultry processing plant

Campylobacter jejuni is worldwide recognized as a human foodborne pathogen. It is widely present in poultry meat and slaughterhouses, but little is known about its fate during the processing of poultry meat preparations. In stress conditions, this pathogen can enter into a viable but non-culturable state, where quantitative PCR (qPCR) becomes more convenient for its detection. In this study, two different pairs of primers, targeting the rpoB and the hipO genes, were compared for its detection and quantification by PCR. Two calibration curves were prepared: one for the meat samples and the other for the environmental samples. rpoB primers showed higher sensitivity with a quantification limit of 1 log cfu/g or ml. Microbial Assessment Scheme (MAS) was used to select the Critical Sampling Locations (CSLs) along the poultry processing line. Forty-six out of 48 samples were positive by qPCR after enrichment (t = 48 ) while only 6 samples were positive by ISO 10272-1:2006. Forty-three samples showed positive signal without enrichment (t = 0 h), however only 16 samples could be quantified. These results showed the high prevalence of C. jejuni in the poultry industry and the need for new, rapid and sensitive techniques, such as qPCR, for the detection and quantification of C. jejuni in meat and environmental samples.     

东北市售鸡肉中空肠弯曲杆菌的分离鉴定、分型及耐药性分析

为了揭示东北地区市售鸡肉中空肠弯曲杆菌的污染情况、种群特征及耐药性。采集东北地区市售鸡肉样品1 000份,分离鉴定空肠弯曲杆菌。通过多位点序列分型技术分析空肠弯曲杆菌的遗传多样性。利用K-B琼脂扩散法检测该致病菌对8种抗生素的耐药性。结果表明:1 000份样品中共分离出62株空肠弯曲杆菌,污染率为6.2%;62株空肠弯曲杆菌被分为14个序列型(Sequence Type,ST),其中ST5和ST1为该种群的优势ST;耐药性分析结果显示,62株空肠弯曲菌对环丙沙星、复方新诺明和头孢噻肟具有高度的敏感性。     

空肠和结肠弯曲菌鞭毛蛋白基因fla A的检测

目的 运用一种快速、敏感、特异的检测空肠和结肠弯曲菌的方法.方法 以空肠和结肠弯曲菌所共有特异的鞭毛蛋白基因 fla A的一段高度保守序列为引物,用PCR法扩增fla A基因上的一段约1 700 bp的片断.用该引物对空肠和结肠弯曲菌的标准株、福建省的食品分离株进行PER扩增检测,并同时检测该PCR方法的敏感性.结果 扩增片断表现出极好的特异性,2株空肠和结肠弯曲菌标准菌株、8株分离自不同食品样品的空肠穹曲菌和结肠弯曲菌菌株均为阳性,且敏感性实验显示该PCR方法的反应体系最低检出菌量为6 CFU.结论 该方法快速、敏感、特异,可用于突发性食物中毒和暴发感染的调查.