Parallel analysis of 7 food-borne pathogens using capillary electrophoresis-based single-strand conformation polymorphism

Capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex polymerase chain reaction (PCR) method was used for the detection of 7 pathogens associated with foodborne illness, including Salmonella enterica, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Vibrio parahaemolyticus, and Escherichia coli O157:H7. The method was applied to model food systems, both of culture medium and cooked rice. The detection limit of individual microbes was in the range of 10¹–10³ CFU/g, and that of the mixture of 7 microbes was 10³ CFU/g in the cooked rice sample. This method allowed the detection and identification of all 7 food-borne pathogens within 5 hr without the requirement for enrichment steps.     

Identification of the fish species of raw or cold-smoked salmon and salmon caviar by single-strand conformation polymorphism (SSCP) analysis

Differentiation between ten salmonid fish species belonging to the genera Salmo, Oncorhynchus and Salvelinus was achieved by single strand conformation polymorphism analysis (SSCP) of PCR products of mitochondrial and nuclear genes. Amplicons (300–460 bp in size) of the genes for cytochrome b, parvalbumin and growth hormone gave species-specific patterns of single-stranded DNA in native polyacrylamide gel electrophoresis (PAGE). The method was successfully used to identify products from raw or cold-smoked salmon, as well as from salmon roe.     

Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods

The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.     

Analysis of ERIC-PCR genomic polymorphism of Salmonella isolates from chicken slaughter line

The chicken slaughter line is a source of cross-contamination of Salmonella. In this study, ERIC-PCR was applied to analyse the ERIC-PCR genomic polymorphism of Salmonella isolates from a commercial chicken slaughter line and to trace the route of contamination. Samples were collected from carcasses and contact surfaces at the points of post-evisceration, post-chilling and post-grading. The prevalence of Salmonella at the evisceration point was high but significantly decreased along the slaughter line. The ERIC-PCR fingerprints indicated that a total of seven groups were clustered, and the genotypic diversity of isolates progressively decreased along the slaughter line. By tracing the genotypic diversity, contact surfaces at post-evisceration were found to be the major contamination sources of Salmonella during chicken processing, since the genotype diversity of isolates from the post-evisceration point could be exactly matched to that from the post-chilling and post-grading points. Interestingly, three Salmonella strains were still detected after decontamination and washing; these three isolates having a strong capacity for attachment were able to produce biofilm on polystyrene surfaces. This study suggests that the evisceration point is the source of cross-contamination, with Salmonella isolates still present after washing procedure. Therefore, more effective measures must be undertaken to control the spread of Salmonella in such processing lines.     

一起伦敦沙门氏菌食物中毒分离株的病原特征分析

目的 分析2020年广州市一起伦敦沙门氏菌食物中毒分离株的病原特征。方法 对分离纯化的10株沙门氏菌进行血清型鉴定,耐药检测,毒力岛(salmonella pathogenicity island,SPI)基因片段SPI-1、SPI-2、SPI-3、SPI-4、SPI-5的PCR检测及脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)分析。结果 10株沙门氏菌血清型鉴定抗原式均为O₁₀：l、v：1、6,即伦敦沙门氏菌,药敏试验显示10株沙门氏菌对氨苄西林(AMP)、萘啶酸(NAL)、头孢唑林(CFZ)、复方磺胺(SXT)、氨苄西林／舒巴坦(AMS)100 %耐药,对头孢噻肟(CTX)、头孢西丁(CFX)、头孢他啶(CAZ)、亚胺培南(IPM)、庆大霉素(GEN)、四环素(TET)、氯霉素(CHL)、环丙沙星(CIP)100 %敏感。毒力岛基因SPI-1~SPI-5全部为阳性。PFGE聚类分析显示,5株病患株、3株食品株、2株环境株为高度同源株。结论 引起此次食物中毒事件的病原菌是伦敦沙门氏菌,应进一步加强餐饮环节的卫生监测和控制。     

Molecular analysis of Salmonella isolates recovered from processed Turkey carcasses

This study was carried out to determine the prevalence of some virulence characteristics associated with Salmonella isolates recovered from processed turkey carcasses in the Midwestern region of the United States. A total of 94 Salmonella isolates recovered from turkey carcasses from two processing plants (A and B) were examined to determine the prevalence of invA, pagC, and spvC genes. Bioassays also were used to evaluate aerobactin and colicin production. All isolates (100%) were positive for the presence of invA and pagC but were negative for spvC. Overall, 19.1% of all isolates tested were positive for aerobactin production, and 25.5% of all isolates were positive for colicin. Aerobactin and colicin production differed among isolates recovered from the two plants; more isolates from plant B produced these compounds. The Salmonella isolates examined in this study possess significant potential for causing human illness.     

16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique

We have developed an analytical technique for the 16S rRNA gene that comprises whole-genome amplification and the polymerase chain reaction (PCR)-minigel-single-strand conformation polymorphism technique (WGA-SSCP). Under optimal conditions, SSCP bands could be detected when genomic DNA from bacteria of interest comprised 0.5% or more of the specimen. This method will be effective for the identification of nonculturable bacteria in a microbial community.     

我国四省肉鸡屠宰厂沙门氏菌脉冲场凝胶电泳分子分型结果分析

目的 了解我国四省肉鸡屠宰厂沙门氏菌脉冲场凝胶电泳分子分型情况。方法 参照美国疾病预防控制机构PulsNet实验方法,对2010年监测四省肉鸡屠宰厂分离到的167株沙门氏菌,运用XbaⅠ酶进行酶切并完成PFGE分析,利用BioNumerics 软件对分离株的指纹图谱进行聚类分析并建立数据库。结果 167株沙门氏菌共分为18个群,包括了75个不同的带型。相同血清型具有相似带型,基本归于同一群。65株印地安纳沙门氏菌分成了38个带型,54株肠炎沙门氏菌分成了16个带型,16株奥尔巴尼沙门氏菌分成1个带型。结论 各省沙门氏菌的带型具有地区性差异, 又具有优势型别的交叉。屠宰厂存在严重的交叉污染,加强加工环节关键控制点沙门氏菌的监测和干预,从而降低肉鸡及其产品中沙门氏菌的污染,这是从源头控制污染的根本措施。     

2016年中国26个省市食源性沙门菌耐药性特征分析

目的 了解2016年中国26个省、直辖市和自治区食源性沙门菌的耐药状况。方法 采用微量肉汤稀释法测定755株食源性沙门菌对10类16种抗生素的药物敏感性,采用实时荧光定量聚合酶链式反应方法检测mcr-1基因的存在情况。结果 72.7%(549/755)的沙门菌对受试的16种抗生素呈现不同程度的耐药性,其中萘啶酸(NAL)、四环素(TET)、氨苄西林(AMP)、氨苄西林/舒巴坦(SAM)4种抗生素的耐药率较高,均在34%以上,未见碳青霉烯类[亚胺培南(IPM)、美罗培南(MEM)]耐药菌株,44.4%(335/755)的沙门菌同时耐受3类或3类以上抗生素,表现为多重耐药,同时耐受抗生素种类最高为8类。共存在134种耐药谱,优势耐药谱型为NAL、TET和AMP-SAM-NAL。全部菌株中检出2株携带mcr-1基因的菌株,分别为八重耐药的德尔卑沙门菌(Salmonella Derby)和七重耐药的鼠伤寒沙门菌(Salmonella Typhimurium)。部分省份沙门菌耐药率较高。结论 2016年中国26个省、直辖市和自治区食源性沙门菌整体耐药水平较高,多重耐药情况严重,我国食源性沙门菌中存在携带mcr-1基因的多重耐药菌株,应引起关注。     

食品沙门氏菌实时荧光PCR快速检测方法建立

根据Genbank提供的沙门氏菌ttrBCA基因序列设计引物和探针,建立了食品沙门氏菌实时荧光PCR快速检测方法。结果显示,该方法只对沙门氏菌基因呈阳性反应,而对其它常见非阳性菌株(志贺氏菌、金黄色葡萄球菌、大肠杆菌、奇异变形杆菌、普通变形杆菌、绿脓杆菌、单增李斯特氏菌、阴沟肠杆菌、副溶血性弧菌)基因组DNA均呈阴性反应,对模拟添加沙门氏菌样品检测,检测低限为240cfu/mL沙门氏菌的DNA,检测食品样品增菌液,仅需约3h,结果表明,该方法适用于食品样品的快速检测。     

TaqMan探针法实时荧光定量PCR快速检测沙门菌的探讨

为建立一种快速、灵敏、特异的实时荧光定量PCR法用于沙门菌的检验，根据GenBank上登录的编号为AE016841的沙门菌序列，应用生物学软件在fimY基因的保守区设计引物和TaqMan探针，同时应用BLAST程序进行网上序列比对，并进行筛选、优化。用鼠伤寒标准菌和60份食品样本进行本检测方法的特异性、敏感性和重复性试验，并与常规法和科玛嘉平板分离法做比较。本方法对沙门菌的检测具高度的特异性，检测的灵敏度这102CFU／ml，从增菌至完成检测仅需24h左右，是一种快速检测沙门菌的敏感、特异的新方法。     

拉曼光谱法快速鉴别劣质火锅油

长期食用劣质火锅油会对人体产生严重伤害。为快速鉴别劣质火锅油,对火锅油和6种普通的食用植物油进行拉曼光谱测试分析。结果表明：火锅油的拉曼光谱在1 150 cm-1和1 525 cm-1处有明显的谱峰,而其他食用植物油则没有,利用主成分分析法可以明显地区别火锅油和普通食用植物油;对同一种火锅油经过反复熬煮和存放,其拉曼光谱在1 525 cm-1谱峰处的相对强度与酸值具有较高的相关性,相关系数达到0.925 6。拉曼光谱法可以作为一种现场快速鉴别火锅油与食用植物油以及检测火锅油质量的潜在方法。     

Restriction fragment length polymorphisms analysis by pulsed-field gel electrophoresis for discrimination of Staphylococcus aureus isolates from foodborne outbreaks

A number of outbreaks of disease due to Staphylococcus aureus occurring in Aichi-ken, Japan, have provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. Coagulase types, enterotoxin types, phage types, and restriction fragment length polymorphisms (RFLPs) as assessed by pulsed-field gel electrophoresis (PFGE) was performed for S. aureus infections diagnosed in the area of Aichi-ken. Among the 56 isolates of S. aureus from 30 outbreaks, 15 distinctive RFLP types were found by digestion with the restriction enzyme, SmaI. A total of 32 isolates from patients, foodstuffs and cooks on six occasions had the same RFLP types, coagulase types, enterotoxin types and phage types in the same outbreaks. Moreover, the coagulase and phage types could be separated in terms of RFLP. In one outbreak, ten isolates, which were derived from six patients, two foodstuffs and two cooks, had the same coagulase type, enterotoxin type, phage type, and RFLP type. This PFGE method may therefore prove useful for subclassifying S. aureus and differentiating isolates of the same coagulase types and phage types derived from sporadic cases and those derived from foodborne outbreaks.     

基于低场核磁共振技术的大米品种快速鉴别

为探索大米无损检测技术,提出了一种基于低场核磁共振技术的快速、无损鉴别大米品种的新方法。以不同地域的大米为低场核磁共振检测对象,利用主成分分析法(PCA)分析处理Carr-Purcell-Meiboom-Gill(CPMG)序列的检测数据。实验结果表明,不同地域的大米在主成分得分图上可以得到很好的区分;说明所提出的方法具有很好的分类和鉴别作用,为大米的品种鉴别提供了一种新方法。     

Antimicrobial susceptibilities and epidemiological analysis of Salmonella enteritidis isolates in Korea by phage typing and pulsed-field gel electrophoresis

A total of 81 isolates of Salmonella Enteritidis were analyzed by antibiotic susceptibility, phage typing, and pulsed-field gel electrophoresis (PFGE). Thirty-two isolates came from broiler carcasses and pig feces, and 49 isolates were from humans in Seoul and suburbs of Seoul, Korea. Antibiotic resistance was most prevalent among human isolates. Of human isolates, 89.8% were resistant to more than two antibiotics, while 64.7% of poultry isolates and 13.3% of pig isolates showed multiple resistance to more than two antibiotics. The most common phage type (PT) was PT1, followed by PT30 or 33, PT21 and PT20a. The isolates showed six PFGE patterns with XbaI or SpeI digestion, and five PFGE patterns with NotI digestion. But a single pattern, PFGE X1, S1, or N1, was predominant and the rest of the PFGE patterns differed by only one or two bands. Results indicated the spread of a genetically related clone of Salmonella Enteritidis in foods and humans in Korea and that phage typing as well as PFGE may offer an improved level of discrimination for the epidemiological investigation of Salmonella Enteritidis.     

Microbial diversity in Tunisian olive fermentation brine as evaluated by small subunit rRNA - Single strand conformation polymorphism analysis

The microbial diversity of a Tunisian olive fermentation brine was analysed using a culture-independent approach based on the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). SSCP patterns show a remarkably simple microbial community but higher for bacterial community than for the eukaryotic community. This study did not show the presence of archaeal populations. After PCR amplification, two small subunit (SSU) rRNA clone libraries of Bacteria and Eucarya populations were established. Three bacteria and only one eukaryotic phylotype were identified. Two dominant bacteria showed 100% phylogenetic similarity to the 16S rRNA sequences of Lactobacillus plantarum and Lactobacillus collinoides and represent 85% of bacterial community. The third bacteria phylotype was phylogenetically close related to the 16S rRNA sequence of a moderately halophilic bacterium belonging to the class gamma Proteobacteria. The dominant eukaryotic phylotype was identified as a Pichia membranaefaciens.     

Analysis of the Salmonella typhimurium isolates from food-poisoning cases by molecular subtyping methods

Salmonella typhimurium is one of the common food pathogens which may cause gastroenteritidis in human. In order to find the prevalent and recirculating strains of S. typhimurium which caused food-poisoning diarrhoea cases in northern Taiwan, 45 randomly selected S. typhimurium strains isolated from food-poisoning cases during 1991–1994 were subjected to pulsed field gel electrophoresis (PFGE), plasmid profile and random amplified polymorphic DNA (RAPD) analysis. Results showed that when genomic DNAs of these Salmonella strains were digested with XbaI, AvrII and SpeI, respectively, followed by PFGE, a total of 26 PFGE patterns combinations were observed. Of them patterns X5A4S4 and X2A2S2 were the major patterns since they contained 13 and four strains, respectively. For plasmid profiles, 21 patterns were found. When plasmid profiles were combined with PFGE patterns, a total of 35 subtypes were found and strains of the subtype of X5A4S4e7 (seven strains) and X2A2S2e7 (three strains) were the prevalent strains. Subtyping by RAPD could not further differentiate these strains grouped by PFGE and plasmid profiles. Since strains of the same patterns combinations were isolated from unrelated food-poisoning cases during 1991–1994 in northern Taiwan, these strains may be the prevalent strains and recirculation of these strains for food-poisoning cases is possible.     

环介导等温扩增技术快速检测沙门菌

环介导等温扩增(loop-mediated isothermal amplification,LAMP)是一种在等温条件下高特异、高效、快速地扩增靶序列的DNA扩增新技术.以沙门菌(Salmonella spp.)为研究对象,根据其特异性的invA基因,设计了一套特异性引物对该基因进行了LAMP,同时优化了其反应条件,建立了沙门菌的LAMP快速检测技术.结果表明,LAMP的最佳反应条件为外引物浓度5 pmol/L、内引物浓度40pmol/L,Mg2 浓度6mmol/L,dNTP浓度0.8mmol/L,甜菜碱浓度0.8mmol/L,Bst DNA聚合酶8u,反应温度63℃,反应时间1 h.在此条件下,LAMP检测沙门菌DNA的敏感度达10fg/反应,且与其他常见的细菌无交叉反应.其对牛奶样品的检出量为102cfu/mL,适合于食品中污染沙门菌的快速检测.     

免疫磁捕获-荧光定量PCR快速检测食品中沙门氏菌

建立了免疫磁珠分离（Immunomagnetic separation,IMS）联合荧光定量PCR（Real-time PCR）技术准确、快速检测食品中沙门氏菌（Salmonella spp）的方法。采用亲和纯化的羊抗沙门氏菌抗体与Dynabeads M-280磁珠制备沙门氏菌免疫磁珠,优化反应条件,建立免疫磁珠分离方法。同时根据沙门氏菌特异性ttr基因,建立Real-time PCR体系,并检测其特异性及敏感性。结果显示,沙门氏菌可产生荧光信号,而其他细菌均未见明显荧光信号。利用所建立的免疫磁捕获-荧光定量PCR（IMS-real-time PCR）方法可以检测初始含菌量最低为6.5CFU/25g的食品样品,全过程需时约8h。150份实际食品样品中,IMS-real-time PCR方法检出27份阳性样品,免疫磁珠联合XLT4平板培养法检出18份阳性样品,传统国标法检出14份阳性样品。