ERIC-PCR和Sau-PCR对单增李斯特菌分型稳定性研究

为了研究肠杆菌间重复序列-聚合酶链式反应(ERIC-PCR)和Sau-PCR两种现代分子分型方法对单增李斯特菌(LM)分型的稳定性,取分离自广州市菜市场、超市及厦门某食品加工厂不同食品来源的5株单增李斯特菌进行实验,分别将这5株单增李斯特菌株进行室温放置培养和传代培养,提取室温放置培养24 h、48 h、和72 h及传代培养至第5代、10代、15代和20代的基因组DNA,然后同时进行ERIC-PCR和Sau-PCR分型,观察随着放置时间延长及传代次数增加其指纹图谱的变化。结果显示,5株单增李斯特菌在室温放置培养及传代培养后除部分条带发生缺失外均未出现条带增加现象,整体条带变化不大,两种分型方法的同源性分别在92%和94%以上,表明ERIC-PCR和Sau-PCR两种分型方法在室温放置培养72 h和传代培养20代内对单增李斯特菌分型相对比较稳定,具有流行病学意义。     

食源性单增李斯特菌的ERIC-PCR和Sau-PCR分型研究

对从广州市天河区超市和菜市场及厦门某食品加工厂分离得到的单增李斯特菌进行ERIC-PCR和Sau-PCR检测,进行溯源研究及遗传多样性分析。ERIC-PCR将22株单增李斯特菌共分为8个基因型,其中以h型为主,共有8株菌。Sau-PCR方法将这批菌分为7个基因型,主要型别为E型和G型,二者共占50%。两种分型方法的结果呈现出一定的差异与关联,使用两种不同分型方法进行综合分析,有助于更好地认识单核细胞增生李斯特菌(LM)菌株间的遗传关系和流行病学特点。     

ERIC-PCR技术对单增李斯特菌的溯源分析

以质控菌株ATCC 19115为对照,采用ERIC-PCR方法对从三个市场猪肉样品分离到的17株单增李斯特菌(Listeria monocytogenes)进行了基因分型,探讨了单增李斯特菌基因型与区域分布及流行性的关联性.结果表明,17株单增李斯特菌菌株可分为六个主要基因类群,其中Ⅳ型菌株最多,为主要污染类群,而这些菌株来自于市场三;市场一和市场二分离到的菌株主要分别为Ⅰ型和Ⅳ型.因此,ERIC-PCR方法适用于对单增李斯特菌的溯源分析和流行病学调查,具有简单、方便、快捷、准确的特点.     

单核细胞增生李斯特菌食品分离株的分子分型鉴定

单核细胞增生李斯特菌是一种重要的食源性条件致病菌,不同亚型菌株的致病力存在较大差异。本文以86株单核细胞增生李斯特菌食品分离株为研究对象,采用多重PCR的血清分型方法 ,将这些分离株分为3个血清组,即:1/2a或3a(72.1%,n=62),1/2b或3b或7(19.8%,n=17),1/2c或3c(8.1%,n=7)。采用ERICPCR亚分型方法 ,将这些分离株和4株标准菌株分为10个类群。对比ERIC-PCR的指纹图谱与血清分型结果 ,两者呈现较高的相关性。将血清分型和ERIC-PCR方法相结合,可实现不同来源单核细胞增生李斯特菌食品分离株的同源性分析。这些分型结果为该菌的流行病学调查提供数据支持。     

产PrtS蛋白酶嗜热链球菌的筛选及其生长特性研究

从商业发酵剂中初步分离出8株疑似嗜热链球菌,与实验室保存的26株嗜热链球菌,共收集到34株菌。通过PCR反应获得具有PrtS基因的一株菌,利用FSDA培养基确定该菌株具有PrtS蛋白酶活性,经生理生化实验和16S rDNA序列同源性分析该菌株为嗜热链球菌,命名为Streptococcus thermophiles 922。该菌株在LM17培养基中培养10 h时,活菌数达到9.68 log cfu/mL;在脱脂乳培养基中,产酸速度快,产酸量较高。实验结果为深入探究PrtS蛋白酶对嗜热链球菌生长和发酵性能影响提供基础。     

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。     

空肠弯曲菌分离株ERIC-PCR分型和生化分型的比较研究

建立空肠弯曲菌(Campylobacter jejuni)的ERIC-PCR分子生物学分型技术,比较ERIC-PCR分型和生化分型的分型效果。从菌株TY1273出发,运用L16(54)正交试验,对Mg2+、dNTPs、引物和TaqDNA聚合酶浓度等因素在较大范围水平内进行反应体系条件摸索,得到一个初步的优化体系;在此基础之上进行单因素的进一步小范围水平内的微调优化,得到最终的优化体系;最后以优化的ERIC-PCR方法对24株C.jejuni分离株分型;同时根据API Campy生化反应结果进行生化分型,比较ERIC-PCR分子分型方法和生化分型方法。结果显示ERIC-PCR方法将24株菌扩增均得到大小在100 bp-3000 bp之间的条带,并可将其分为22个基因型,分辨系数为0.92,具有较高的分辨力。生化分型将24株菌分为19个生化型,显示了菌株基因的多样性。表明ERIC-PCR技术比生化分型能更好的体现菌株的遗传多样性,且具有简便和分辨力搞等优点,可用于C.jejuni的多样性研究。     

食品中单增李斯特菌的血清分型及脉冲场凝胶电泳分型

对进口食品和国内食品中的单增李斯特菌进行血清学分型和分子分型研究,探讨不同来源菌株之间的遗传相关性及不同分型方法之间的联系。对分离的单增李斯特菌进行血清学分型和脉冲场凝胶电泳分型。69株单增李斯特菌分为4种血清型,主要血清型为1/2a(3a)型,脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)分型分为55种带型,主要型别为GX6A12.CN0018。食品中的单增李斯特菌分子型别呈现多态性,进口食品分离株和国内食品分离株之间无相关性。     

副溶血性弧菌ERIC-PCR分型及毒力基因检测研究

目的:建立副溶血性弧菌ERIC-PCR分子分型技术,分析副溶血性弧菌标准菌株及分离株基因组DNA ERIC-PCR指纹图谱,并对副溶血性弧菌毒力基因进行检测,以了解不同来源副溶血性弧菌毒力基因携带情况.方法:提取副溶血性弧菌基因组DNA,以肠杆菌基因间共有重复序列(ERIC)为引物进行PCR扩增,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析,并以相似性系数构建聚类图;通过PCR方法对直接耐热溶血素(TDH)和耐热直接相关溶血素(TRH)进行检测.结果:26株副溶血性弧菌均可扩增产生可重复的DNA指纹图谱,ERIC-PCR可将26株菌分为12个型,分辨力指数为0.926;只在临床分离株中检测到TDH基因,而除一株标准菌株外,所有菌株都未检测到TRH基因.结论:研究显示ERIC-PCR可从分子水平对副溶血性弧菌基因组DNA进行快速指纹图谱分析,同时结合毒力基因检测,能够为副溶血性弧菌食物中毒疾病的预防和流行病学调查提供科学依据.     

温州市单核细胞增生李斯特菌的毒力基因及血清学分型和分子分型研究

目的了解温州市近十年单核细胞增生李斯特菌分离株的血清型、毒力基因及分子分型特征。方法用聚合酶链式反应(PCR)方法对单核细胞增生李斯特菌进行血清型及毒力基因检测;用多位点序列分型(MLST)方法对单核细胞增生李斯特菌进行分子分型,并绘制MLST数据的最小生成树。结果 97株单核细胞增生李斯特菌分离株分为4种血清型,以血清型1/2b、1/2a为优势血清型,占比分别为48.45%(47/97)、35.05%(34/97);而毒力基因iap、prfA基因阳性率均为100.00%(97/97),hlyA、inlA基因阳性率均为97.94%(95/97),plcB基因阳性率为96.91%(94/97)。其中患者分离株5种毒力基因阳性率均为100.00%(6/6)。97株单核细胞增生李斯特菌分离株得到20个MLST型别,其中ST87型是优势型别,其次为ST121和ST9,ST1和ST779型是患者特有的,ST2、ST3、ST5型分布于食品和患者分离株。结论温州市不同来源的单核细胞增生李斯特菌分离株分子型别呈多态性,食品和患者分离株存在相同的ST型,且这些菌株大部分携带毒力基因,具有潜在的致病性,因此食品中单核细胞增生李斯特菌污染的潜在风险不容忽视。     

分型方法在单核细胞增多性李斯特菌监测溯源中的应用研究进展

单增李斯特菌(LM)是引起人和动物李斯特菌病的重要食源性病原菌,具有较高的病死率,严重危害人类公共卫生安全和畜牧业发展。快速准确的鉴定LM污染来源,对有效控制李斯特菌病的暴发和蔓延发挥重要作用。建立高效快捷的分型方法是LM溯源的关键。目前LM分型方法主要分为表型分型和分子分型方法,每种分型方法各有优劣,具有适用不同的流行病学调查范围。本文就LM分型方法的研究进展进行综述,以期为开展LM的暴发确认和溯源检测方法的选择提供参考依据,对防御并控制LM引起的食源性疾病的暴发和传播具有重要意义。     

COMPETITION OF FOOD BACTERIA WITH LISTERIA MONOCYTOGENES DURING ENRICHMENT CULTURE

Thirty-two foodborne bacterial isolates were tested as potential competitors of Listeria monocytogenes strain LM82 during enrichment because of their resistance to the selective agents in Listeria enrichment and isolation media. Competitive ability of each isolate was classified as weak, moderate, or strong by determining the ratio at which it masked identification of LM82 at an inoculation concentration of 10 colony forming units (CFU)/10 mL of Listeria enrichment broth. Of the competitive isolates identified, six were Enterococcus spp., two were Staphylococcus spp., and one was a Corynebacterium sp. Although several strains of Enterococcus faecium were examined, not all were competitive. Of six other bacterial strains associated with food fermentations and tested for competitiveness with LM82, one, a Gram-positive tetrad, was competitive. This study showed that although food microfloral strains that are able to survive in enrichment and isolation environments are fairly common, they do not necessarily compete with Listeria. Not all strains in a competitive species are necessarily competitive .     

河北地区食源性单核细胞增生李斯特菌脂肪酸的分型研究

对脂肪酸分型方法在单核细胞增生李斯特菌(LM)菌株鉴定、菌株相似性分析等方面的应用价值进行评价。本研究选取2005~2007年从河北地区六大类食品中分离到的90株LM菌,提取脂肪酸,利用MIDI公司Sherlock系统进行菌体脂肪酸成分分析,使用SPSS 19.0软件对获得的数据资料进行统计分析及聚类分型,并将脂肪酸分型与传统的血清分型和分型金标准PFGE分型进行比较。结果表明,脂肪酸分析法判定LM菌的符合率为96.67%,所有菌株共检出20种脂肪酸成分,主要脂肪酸成分有3种,分别为脂肪酸15:0anteiso、17:0 anteiso和15:0 iso。各血清型间脂肪酸含量存在一定差异,血清1/2c型菌株与血清1/2a、1/2b和4b型菌株相比,有2种主要脂肪酸含量差异有统计学意义(P0.01)。与PFGE分型相比,在对结构简单的小样本资料的菌株亲缘关系鉴定中脂肪酸分型更具优势。将脂肪酸分型与血清学分型和PFGE分型相结合能够更好的分析LM菌菌株之间的相关性。     

Molecular ecology of Listeria monocytogenes and other Listeria species in small and very small ready-to-eat meat processing plants

A longitudinal study was conducted to track Listeria contamination patterns in ready-to-eat meats from six small or very small meat processing plants located in three states over 1 year. A total of 688 environmental sponge samples were collected from nonfood contact surfaces during bimonthly visits to each plant. Overall, L. monocytogenes was isolated from 42 (6.1%) environmental samples, and its prevalence ranged from 1.7 to 10.8% across different plants. Listeria spp., other than L. monocytogenes, were isolated from 9.5% of samples overall, with the prevalence ranging from 1.5 to 18.3% across different plants. The prevalence of L. monocytogenes correlated well with that of other Listeria spp. for some but not all plants. One L. monocytogenes isolate representing each positive sample was characterized by molecular serotyping, EcoRI ribotyping, and pulsed-field gel electrophoresis typing. Seven sample sites tested positive for L. monocytogenes on more than one occasion, and the same ribotype was detected more than once at five of these sites. Partial sigB sequencing was used to speciate other Listeria spp. isolates and assign an allelic type to each isolate. Other Listeria spp. were isolated more than once from 14 sample sites, and the same sigB allelic type was recovered at least twice from seven of these sites. One plant was colonized by an atypical hemolytic L. innocua strain. Our findings indicate that small and very small meat processing plants that produce ready-to-eat meat products are characterized by a varied prevalence of Listeria, inconsistent correlation between contamination by L. monocytogenes and other Listeria spp., and a unique Listeria molecular ecology.     

Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB

A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters. Four sets of oligonucleotide primers were evaluated. The set targeting a 902-bp region of the inlAB gene was the most specific. This PCR product was detected in 51 L. monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected in other Listeria spp. (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L monocytogenes. The detection limit of the PCR assay was 10(5) CFU per ml of pure cell culture. However, the assay could detect as few as 10(1) CFU of L. monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30 degrees C. The total assay time including enrichment was approximately 24 h. These results suggest that the PCR assay can be used to rapidly detect L. monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.     

A comparative study of different PCR-based DNA fingerprinting techniques for typing of lactic acid bacteria

A set of 92 lactic acid bacteria strains and 10 type/reference strains was analysed using three molecular techniques (randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), enterobacterial repetitive intergenic consensus (ERIC-PCR) and the polytrinucleotide (GTG)₅-PCR), in order to compare their discriminatory ability for typing of these bacteria. The results indicated that RAPD-PCR generated patterns with the largest number of bands, adequately clustering the isolates belonging to the same genus. Likewise, it showed the highest discriminatory capacity, while no discrimination between isolates of different genera was possible with ERIC-PCR and (GTG)₅-PCR. Therefore, the use of RAPD-PCR has turned out to be the most suitable procedure for typing LAB isolates belonging to different genera and species obtained from some fermented foods such as goat and Manchego cheeses, Almagro eggplant fermentations and Tempranillo wines.     

Listeria monocytogenes in pork slaughtering and cutting plants. Use of RAPD, PFGE and PCR-REA for tracing and molecular epidemiology

In order to determine the origin of pork cuts contamination by Listeria monocytogenes, 287 isolates, collected from five French pork slaughtering and cutting plants, from live pigs to pork cuts, were characterised using three molecular typing methods: random amplification of polymorphic DNA (RAPD) carried out with five different primers, genomic macrorestriction using ApaI with pulsed-field gel electrophoresis (PFGE) and a PCR-restriction enzyme analysis (PCR-REA) based on the polymorphism existing within the inlA and inlB genes. Results obtained from RAPD and PFGE were closely related and distinguished respectively 17 RAPD types (r1-r17) and 17 PFGE types (a1-a17) among the 287 isolates, whereas the PCR-REA analysis only yielded two profiles (p1 and p2). Considering the combined results obtained with the three molecular typing methods, 19 Listeria monocytogenes genotypes (1-19) were distinguished. Serotyping led at least four serotypes being distinguished: 1/2a, 3a, 1/2c and 3c. The application of genotyping identified the predominance of a Listeria monocytogenes strain of type (1) and other very closely related ones (5, 9, 10, 12, 13, 14, 16 and 19) which were present on pork as well as in the environment within the five investigated plants. This study also pointed out the presence of these closely related Listeria monocytogenes strains over a 1-year period in the environments of two plants, even after cleaning and disinfection procedures. This highlights the possibility for some Listeria monocytogenes strains to persist in pork processing environments and raises the problem of the efficiency of cleaning and disinfection procedures used in pork slaughterhouses, chilling and cutting rooms.     

1例急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病原学及分子分型特征分析

目的对北京市1名急性淋巴细胞白血病患者感染单核细胞增生李斯特菌病例进行病因溯源,对分离到的单核细胞增生李斯特菌进行血清学分型、耐药及分子分型研究。方法对患者不同时期外周血分离的2株单核细胞增生李斯特菌和1株环境涂抹样品单核细胞增生李斯特菌分离株进行血清学分型、耐药性分析、脉冲场凝胶电泳(PFGE)和多位点序列分析(MLST)。结果 3株单核细胞增生李斯特菌均为1/2a-3a血清型,耐药结果一致,均对青霉素、氨苄西林、复方新诺明、美罗培南及红霉素敏感,3株菌的PFGE带型一致,MLST型别均为ST155。结论本研究中患者生活环境中存在单核细胞增生李斯特菌的污染情况,高度怀疑患者感染单核细胞增生李斯特菌与其生活环境中分离到的菌株为同一来源。