丹东市熟肉制品中食源性致病菌污染状况的调查研究

为了解丹东市熟肉制品中沙门菌、金黄色葡萄球菌和单核细胞增生性李斯特菌的污染及菌相分布状况,为丹东市熟肉制品中致病菌污染提供本地资料,以及熟肉制品卫生监督提供科学依据。依据国标方法以及采用全自动酶联免疫荧光快速筛选仪进行筛选和BBL Crystal细菌鉴定仪鉴定,对130份样品分别进行上述致病菌分离、血清学和生化鉴定。结果共检出致病菌15株,总检出率为11．5％,酱卤类检出率为14．0％、烧烤类检出率为7．5％、白切类检出率为12．5％,其中沙门菌4株、单核细胞增生性李斯特菌4株和金黄色葡萄球菌7株。说明丹东市熟肉制品中存在食源性致病菌污染,其中金黄色葡萄球菌污染最严重。     

FTA滤膜用于PCR检测肉中的金黄色葡萄球菌

利用FTA滤膜,采用PCR技术可直接检测肉及肉制品中的金黄色葡萄球菌,无需增菌,灵敏度高。在采用浮选与溶剂萃取相结合方法的基础上,使用FTA膜可高效地从鲜肉中提取金黄色葡萄球菌的DNA,消除PCR反应的抑制因子。以金黄色葡萄球菌耐热核酸酶基因（nuc）为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。使用FTA滤膜处理样品,再通过PCR方法检测金黄色葡萄球菌,猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为10cfu/mL,可在6h内完成对肉中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h 。实际检测了72份样品,同时与GB 4789.10-94方法及两种快速检测致病性金黄色葡萄球菌检测方法做比较,PCR方法的检出率为72.2％,检出时间为6h,GB 4789.10-94方法检出率为70.8%,检出时间为5d,Prfilm RSA方法的检出率为61.1％, 检出时间为18h,BairdParker R.P.F方法的检出率为69.4％,检出时间为18h,结果表明FTA滤膜用于PCR检测肉中金黄色葡萄球菌检出率高,耗时短。使用FTA滤膜法制备模板DNA,为食品中的致病菌快速检测构建了一个技术平台。     

藏绵羊子宫内膜炎主要致病菌多重PCR检测方法的建立与评价

本研究旨在建立一种多重PCR方法检测青海藏绵羊子宫内膜炎主要的病原菌。首先,提取5种标准菌株基因组,筛选出特异性引物;然后以标准菌株的基因组为模板,建立多重PCR方法。用无菌棉拭子涂抹藏绵羊子宫,置于LB培养液中培养并编号,48 h后提取样品基因组。运用单一PCR法对600份样品基因组进行检测,记录阳性样品;再挑取单一PCR法检测的阳性样品进行多重PCR检测,再次记录阳性样品,通过计算两种检测方法的符合率验证多重PCR方法;随机挑出30份阳性样品,进行病原菌分离鉴定菌种种类。单一PCR检测的样品中,无乳链球菌感染比例占47.33%,大肠杆菌占34.83%,金黄色葡萄球菌占6.5%,未检出沙门氏菌和化脓隐秘杆菌;多重PCR检测的阳性样品中,无乳链球菌感染比例占45.50%,大肠杆菌占33.50%,金黄色葡萄球菌占6.5%;两种检测结果相比较,多重PCR检测出的符合率均高于95%;分离鉴定的病原菌与两种PCR方法检测出的菌种结果基本一致。成功建立了多重PCR方法并检测出引起青海藏绵羊子宫内膜炎的主要病原菌为无乳链球菌、大肠杆菌和金黄色葡萄球菌。     

PCR技术在检测鼠金黄色葡萄球菌中的应用研究

目的　建立实验大小鼠金黄色葡萄球菌的快速检测方法———PCR法。方法　根据已公布的金黄色葡萄球菌耐热核酸酶nuc基因的序列 ,设计并合成一对特异性的引物 ,利用PCR技术扩增nuc基因片段。对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株抽提的DNA进行扩增。结果　金黄色葡萄球菌PCR产物出现 6 6 8bp的特异性DNA扩增片段 ,而其他非金黄色葡萄球菌未出现扩增片段 ,证实了合成的引物对金黄色葡萄球菌具有特异性。将抽提的金黄色葡萄球菌DNA进行系列稀释 ,测定此PCR体系的敏感性 ,结果显示 ,该PCR体系能检出 3pg金黄色葡萄球菌DNA ,且从抽提DNA到PCR扩增及电泳结束仅需 4h。结论　本研究所建立的扩增耐热核酸酶nuc基因检测鼠金黄色葡萄球菌的PCR方法 ,具有快速、可靠、敏感和特异的特点 ,可用于临床样品和金黄色葡萄球菌感染时的检测 ,适合应用于实验大小鼠的监测。     

2010—2015年酒泉市市售食品中食源性致病菌监测与分析

目的：了解甘肃省酒泉市市售食品食源性致病菌的污染状况,为预防与控制食源性疾病发生提供科学依据。方法：按照《2010—2015年食源性致病菌监测计划实施方案》的要求,对2010—2015年酒泉市监测的20类1 575份样品的食源性致病菌监测数据进行整理分析。 结果：共检测样品1 575份,检出阳性菌株196株,总检出率为1116%。各种致病菌的检出率分别为沙门氏菌102 %、蜡样芽孢杆菌1404%、金黄色葡萄球菌395 %、单核细胞增生李斯特氏菌104 %、阪崎肠杆菌118 %、致泻性大肠埃希氏菌090%、副溶血性弧菌500%、创伤弧菌1000%、弯曲菌000%。不同类别食品中,生禽肉、婴幼儿配方食品、生奶、餐饮食品、生畜肉、豆制品、水产品食源性致病菌的污染率较高。生产加工和流通环节是致病菌的主要污染环节。结论：酒泉市市售部分食品存在食源性致病菌污染,食品安全监管部门应加强日常监管,确保人民群众的食品安全。     

PCR技术检测食品中金黄色葡萄球菌肠毒素B基因

目的:金黄色葡萄球菌B型肠毒素是污染食品引起食物中毒的主要原因之一,针对进出口食品卫生监测的需要,研究一种简便、快速、准确的实验方法.方法:利用聚合酶链反应技术(PCR)采用特异的模板探针引物进行杂交,最后通过电泳技术与阳性对照进行比对,来判断阴阳性结果.结果:本方法检出率高,每克样品中有4个金黄色葡萄球菌即可检出,24小时即可报告结果.结论:PCR方法检测食品中金黄色葡萄球菌肠毒素B基因快速、准确,检测周期短,既可提高检出率,又可节省检测时间.     

我国部分地区即食食品和蔬菜中金黄色葡萄球菌污染分布及耐药和基因分型情况

【目的】系统调查了我国15个代表性城市在即食食品(卤肉、烤肉、凉拌菜和巴氏奶)和蔬菜样品中金黄色葡萄球菌(Staphylococcus aureus)的污染分布情况,并对分离株进行耐药性分析及多位点序列分型(Multilocus sequence typing,MLST)研究,为食源性金黄色葡萄球菌的风险识别和分子溯源提供基础数据。【方法】依据GB 4789.10-2010对540份即食食品和蔬菜样品进行定性和最大可能数(Most probable number,MPN)分析;采用K-B纸片扩散法检测金黄色葡萄球菌分离株的耐药特征并通过PCR检测mecA基因;应用MLST方法确证金黄色葡萄球菌的主要ST型。【结果】结果发现9.3%(50/540)的样品中检出金黄色葡萄球菌,其中卤肉污染率最高,为16.3%(30/184),其次为烤肉(9.2%,6/65),蔬菜(6/150,4.0%)污染率最低。定量分析发现62.0%的阳性样品污染水平处于0.3–1.0 MPN/g,其中3份阳性样品污染水平≥110 MPN/g。对50株分离株进行24种抗生素耐药性检测,发现82.0%的分离株对氨苄西林和青霉素耐药,64.0%的分离株为多重耐药株。对mecA阳性分离株进行SCCmec分型,发现均为SCCmecⅣa。所有分离株进行MLST分型,共检出14种型别,其中ST3595和ST3847为新ST型。【结论】普遍的多重耐药性表明我国食源性金黄色葡萄球菌的耐药状况已较为严重,对消费者的安全健康存在潜在威胁。ST型与耐药存在一定的关联性,这为进一步了解该菌在我国食品中的流行趋势和风险评估提供了科学的数据支撑。     

滨州市餐饮服务食品食源性致病菌污染状况

目的：了解滨州市餐饮服务食品中食源性致病菌污染状况,为食源性疾病的防控提供科学依据。方法：按照相关国标和标准检测方法对2014—2017年餐饮服务食品中主要食源性致病菌进行监测。共采集滨州市10大类食品共计328份,进行4种食源性致病菌的分离鉴定。结果：食源性致病菌检测中,328份样品中共检出致病菌45份,总检出率为13.72%。单核细胞增生李斯特氏菌合格率为100.00%,蜡样芽孢杆菌检出率较高。食源性致病菌高检出率样品集中在街头摊点及第三季度。结论：滨州市餐饮服务食品中食源性致病菌存在一定程度污染,应加强餐饮服务环节中食源性致病菌的防控措施,防止食源性疾病的发生。     

化妆品中金黄色葡萄球菌检验能力验证方法及分析

文章依据《化妆品安全技术规范》2015年版和《NIFDC-PT-188化妆品金黄色葡萄球菌检验能力验证作业指导书》，分别对编号为TC1880001和TC18801010的两份样品中的金黄色葡萄球菌进行定性检测，并结合使用BAX?system Q7系统对增菌液进行初步筛查，同时使用VITEK 2COMPACT全自动细菌鉴定系统对分离出的菌株加以鉴定，试图通过外部能力验证，提高对化妆品中金黄色葡萄球菌的检验能力。最终结果表明编号为TC1880001和TC18801010的两个样品中金黄色葡萄球菌检验结果均为阴性。笔者对此次能力验证结果较为满意，并表示检测化妆品中金黄色葡萄球菌时具备常规法和实时荧光PCR法两种能力，能满足日常对化妆品金黄色葡萄球菌的检测监管需求。     

基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。     

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

TEMPO/STA法在水产品金黄色葡萄球菌计数检测中的应用

金黄色葡萄球菌能够引起细菌性食物中毒,其对水产品的污染将严重影响到食品安全和水产品加工出口贸易。本研究使用TEMPO/STA法和PetrifilmTM测试片法对535个出口水产品样品进行检测,并将检测结果进行比较。结果表明,TEMPO/STA法与PetrifilmTM测试片法一致性较好(符合率96.6%,准确度无差异),并具有操作简单、快速、准确和人为误差小的特点。     

一种鉴别临床常见致病性葡萄球菌方法的建立与初步评价

目的建立一种鉴别临床常见致病性葡萄球菌的聚合酶链反应-限制性片段长度多态性（PCRRFLP）方法。方法采用经全自动微生物鉴定系统和分子生物学方法准确鉴定的金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌各3株,提取细菌DNA,PCR扩增tuf基因,扩增产物Alu I、Hinf I双酶切后进行琼脂糖凝胶电泳,分析不同葡萄球菌酶切后带型的差异。收集临床分离葡萄球菌142株,采用建立的PCRRFLP对其进行分类鉴别,随机选择分类鉴别的葡萄球菌各20株,PCR扩增16S r DNA,扩增产物测序,将结果与Gen Bank数据库进行比对,初步评价该方法的准确性。结果金黄色葡萄球菌,表皮葡萄球菌,溶血葡萄球菌均能扩增出长668 bp DNA片段。扩增产物经Alu I、Hinf I双酶切后电泳条带不同,金黄色葡萄球菌出现三条带（108 bp/192 bp/217 bp）,表皮葡萄球菌出现两条带（192 bp/304 bp）,溶血葡萄球菌出现两条带（192 bp/217 bp）。PCR-RFLP结果显示,142株葡萄球菌中金黄色葡葡球菌、表皮葡葡球菌和溶血葡葡球菌分别为67、29和46株。随机挑选的20株不同种葡萄球菌16S r DNA测序结果与Gen Bank数据库对应序列的相似性均〉99%,说明建立的PCR-RFLP方法能准确区分三种常见葡萄球菌。结论 PCRRFLP能准确鉴别临床常见的致病性葡萄球菌,为葡萄球菌病的分子诊断奠定了基础。     

安徽不同地区奶站牛奶中金黄色葡萄球菌及其肠毒素污染状况评估

目的评定安徽地区各奶站牛奶中金黄色葡萄球菌以及肠毒素的污染情况。方法通过从安徽省不同地区30所奶站采集乳样,进行乳源性金黄色葡萄球菌的分离与生化鉴定,并采用PCR技术对分离出的菌株进行金黄色葡萄球菌肠毒素血清型鉴定。结果安徽省30个奶站中有4个地区奶站的牛奶中污染金黄色葡萄球菌;从污染牛奶中共分离出5株金黄色葡萄球菌,检出率为16.7%。经鉴定,所分离出的金黄色葡萄球菌中2株为肠毒素A型,1株为肠毒素C型,2株为同时产肠毒素A和肠毒素C。结论安徽省不同地区奶站中的牛奶污染的金黄色葡萄球菌产肠毒素类型以肠毒素A为主。     

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.     

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.     

Effect of a traditional processing method on the chemical composition of local white lupin (Lupinus albus L.) seed in North-Western Ethiopia

The effect of a traditional Ethiopian lupin processing method on the chemical composition of lupin seed samples was studied. Two sampling districts, namely Mecha and Sekela, representing the mid- and high-altitude areas of north-western Ethiopia, respectively, were randomly selected. Different types of traditionally processed and marketed lupin seed samples (raw, roasted, and finished) were collected in six replications from each district. Raw samples are unprocessed, and roasted samples are roasted using firewood. Finished samples are those ready for human consumption as snack. Thousand seed weight for raw and roasted samples within a study district was similar (P > 0.05), but it was lower (P < 0.01) for finished samples compared to raw and roasted samples. The crude fibre content of finished lupin seed sample from Mecha was lower (P < 0.01) than that of raw and roasted samples. However, the different lupin samples from Sekela had similar crude fibre content (P > 0.05). The crude protein and crude fat contents of finished samples within a study district were higher (P < 0.01) than those of raw and roasted samples, respectively. Roasting had no effect on the crude protein content of lupin seed samples. The crude ash content of raw and roasted lupin samples within a study district was higher (P < 0.01) than that of finished lupin samples of the respective study districts. The content of quinolizidine alkaloids of finished lupin samples was lower than that of raw and roasted samples. There was also an interaction effect between location and lupin sample type. The traditional processing method of lupin seeds in Ethiopia has a positive contribution improving the crude protein and crude fat content, and lowering the alkaloid content of the finished product. The study showed the possibility of adopting the traditional processing method to process bitter white lupin for the use as protein supplement in livestock feed in Ethiopia, but further work has to be done on the processing method and animal evaluation.