RT-PCR检测金黄色葡萄球菌

目的探讨检测金黄色葡萄球菌及其活菌的RT-PCR方法。方法用RT-PCR方法对金黄色葡萄球菌的spa基因进行检测,并做灵敏度和特异性测定,用RT-PCR检测细菌灭活前后的spa基因。结果用spa基因检测金黄色葡萄球菌灵敏度为1.5×104CFU/mL;Spa引物能特异性扩增出金黄色葡萄球菌的标准株和14株临床株的目的片段,对大肠埃希菌、铜绿假单胞菌、表皮葡萄球菌和化脓性链球菌则无特异性扩增条带,而对白色念珠菌有较弱条带扩出;细菌灭活前可以检测出目的基因,灭活后4℃放置24、48和72 h均无目的基因片段扩出。结论可以用spa基因对金黄色葡萄球菌进行活菌检测。     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L,Mg^2＋浓度2．4mmol／L,dNTP浓度2001μmol／L,Taq DNA聚合酶1.5u,退火温度55．0℃-57．4℃之间;在此条件下多重PCR同时检测DNA的敏感性分别是10．2pg、10．2pg、102．0pg,检测时间4h。建立的多重PCR是一种敏感、特异、准确、快速的方法,为同时检测食品中沙门菌、大肠杆菌和金黄色葡萄球菌奠定了基础。     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测*

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol/L、40nmol/L、80nmol/L,Mg²⁺浓度2.4mmol/L,dNTP浓度200μmol/L,TaqDNA聚合酶1.5U,退火温度55.0℃~57.4℃之间;在此条件下多重PCR同时检测DNA的敏     

一株金黄色葡萄球菌小菌落突变株的分离鉴定

摘要：【目的】由于金黄色葡萄球菌（金葡菌）小菌落突变株（small colony variants,简称SCVs ）可引起持续复发性感染,且对氨基糖苷类有抗药性,在临床诊断和治疗上造成很大的困扰。我国国内尚无金葡菌SCVs的报道,本研究旨在分离鉴定出金葡菌SCVs菌株,为国内进行SCVs的相关研究提供生物学材料。【方法】通过细菌的形态鉴定、种特异性基因（nuc）的PCR扩增鉴定以及系列生化实验,从人源、动物源及环境源共104株金葡菌分离株中筛选得到金葡菌SCVs,并通过甲萘醌、硫胺素、胸腺嘧啶和血红素等补     

多重PCR快速检测化妆品中三种致病菌的研究

利用多重PCR技术建立快速检测化妆品中三种致病菌的方法。根据已报道的大肠杆菌phoA基因、铜绿假单胞菌外膜蛋白基因oprL和金黄色葡萄球菌特异性序列SmaI选择特异性引物,对人工染菌化妆品进行多重PCR检测。结果显示,三种致病菌的基因组DNA均可与各自引物特异性结合,扩增产物大小分别为622 bp、504 bp和426 bp。该方法用于人工污染的化妆品中,大肠杆菌的检出限浓度为103 CFU/mL,铜绿假单胞菌和金黄色葡萄球菌的检出限浓度为105 CFU/mL。作者建立的多重PCR方法可同时快速、特异地对化妆品中三种致病菌进行检测,在化妆品行业具有较大的应用价值。     

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。     

金黄色葡萄球菌凝集因子A的免疫原性

为研究金黄色葡萄球菌(Staphylococcus aureus)凝集因子A(ClfA)免疫原性及免疫保护作用,应用PCR方法扩增出金黄色葡萄球菌Newman、Wood46和HLJ23-1株的clfa基因并进行序列分析,再将Newman株的clfa基因插入到pQE-30载体上,导入宿主菌Escherichia coli M15(pREP4)并诱导表达和纯化ClfA重组蛋白。用纯化的ClfA免疫小鼠,检测血清中抗体和细胞因子水平,首次免疫后35 d时用金黄色葡萄球菌Wood46、HLJ23-1、Newman株对小鼠攻毒。结果发现:clfa基因序列高度保守;ClfA重组蛋白在E.coli M15中获得表达;在首次免疫后35 d时血清抗体效价和细胞因子浓度与对照组相比,均显著升高(P<0.05);攻毒结果为蛋白免疫组小鼠获得一定的免疫保护。由此表明,ClfA重组蛋白有较好的免疫原性和免疫保护力。     

长沙某医院产质粒AmpC酶型肺炎克雷伯菌的多重PCR检测与分析

为了了解湖南长沙某医院临床分离的肺炎克雷伯菌中质粒介导AmpC β-内酰胺酶的产生情况及其基因型,收集了该医院2008年3月至2010年10月临床分离的多重耐药肺炎克雷伯菌104株,用头孢西丁纸片扩散法对这些菌株进行表型初筛,用多重PCR确定ampC耐药基因型;结果发现其中有19株对头孢西丁纸片不敏感,疑为产AmpC酶菌株;再经多重PCR扩增,有12株菌分别在约400 bp（11株）和约350 bp（1株）出现了阳性条带,特异性PCR证明此12株菌分别携带了DHA型（11株）和ACC型（1株）ampC耐药基因;产质粒介导AmpC酶肺炎克雷伯菌的分离率为11.5%（12/104）。该医院产质粒介导AmpC酶肺炎克雷伯菌的分离率较高,应对其检测与监测给予足够重视,以指导临床合理选用抗菌药物。     

沙门菌、志贺菌、副溶血性弧菌多重PCR检测方法的研究

建立快速检测沙门菌、志贺菌和副溶血性弧菌的多重PCR方法[1-4].根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌TDH基因设计特异性PCR引物[5-6],被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物.在580、423和245 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现.敏感性试验显示沙门菌在模拟标本中的检测灵敏度为101-2cfu/mL、志贺菌为101cfu/mL、副溶血性弧菌为102cfu/mL.该方法操作方便、分析时间短、特异性和灵敏度高,可用于公共卫生突发事件食源性病原菌的快速检测.     

肺炎链球菌自溶素和溶血素基因的PCR法鉴定

肺炎链球菌是一种致病率和致死率很高的病原菌 ,若无丰富临床检测经验从临床标准中分离鉴定此菌较困难 ,本实验以寡核苷酸引物YH1 YH2、YH7 YH8分别扩增肺炎链球菌自溶素和溶血素基因的 35 4bp和 30 7bp的DNA片段 ,通过改变各种反应条件 ,建立了这两种病原因子基因的PCR检测方法。用此方法对 2 0株肺炎链球菌标准菌株及 7株对照菌株进行了鉴定 ;其扩增产物分别经限制性内切酶TthHB81和和AccI进行酶切以确认扩增产物是否正确 ;用酚 氯仿抽提纯化的全细胞DNA对PCR方法的检测灵敏度进行了测定 ;并利用此方法对 2 8份临床标本分离物进行了鉴定。结果　所有 2 0株肺炎链球菌均可分别用引物YH1 YH2、YH7 YH8扩增出 35 4bp和 30 7bp的DNA片段 ,而对照菌株均呈阴性 ;自溶素及溶血素基因的扩增产物分别经限制性内切酶TthHB81和AccI消化后产生的片段和预期的完全一致 ;两对产物均可从 10fg的全细胞DNA中扩增出目的DNA片段。所建立的两套PCR系统对 2 8份临床标本分离物进行鉴定 ,其中PCR阳性的 15份分离物经生化学特性检查被鉴定为肺炎链球菌。本试验所建立的两套PCR检测系统具有特异性强 ,灵敏度高及操作简单等优点 ,均可用于肺炎链球菌的鉴定。     

奶牛源MRSA肠毒素基因、耐药基因及分子分型分析

为了解宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌的肠毒素基因和耐药基因分布及其分子流行病学特征,本研究通过聚合酶链式反应(polymerase chain reaction, PCR)技术对前期分离于宁夏地区的9株奶牛源耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)进行了18种肠毒素基因和16种耐药基因的检测,同时采用脉冲场凝胶电泳(pulsed-field gel electrophoresis, PFGE)、正向重复序列(direct-repeat unit, dru)和辅助基因调节因子(accessory gene regulator, agr)分子分型技术对MRSA菌株进行分型研究。结果显示所有MRSA菌株均携带经典型肠毒素基因和新型肠毒素基因,共检出12种肠毒素基因,其中selk基因的检出率最高,达到了100%,未检出see、selj、selo、selp、ser和selu基因;11种耐药基因被检出,其中norA、gyrA、grlA和blaZ 4种基因的检出率均达到了100%,未检出tet (O)、optrA、Lin (A)、fexA和cfr基因。PFGE分型结果显示受试菌株间亲缘关系较近;dru分型检出dt11v和dt10a两种型,其中以dt11v(77.8%, 7/9)为主;agr分型主要为agr-Ⅰ型(88.9%, 8/9),agr-Ⅱ型仅有1株。研究表明宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌(MRSA)中的肠毒素基因和耐药基因分布广泛,菌株间亲缘关系较近,agr-Ⅰ-dt11v为MRSA菌株中的流行基因型。这为以后宁夏地区奶牛源MRSA的产毒性、耐药性和分子流行病学特征的进一步研究提供理论依据。     

临沂市奶牛乳房炎病原菌的分离鉴定与药敏试验

目的为弄清临沂市奶牛乳房炎主要病原菌种类及其药物敏感情况,为科学防治本病提供依据。方法采集患乳房炎奶牛的乳样进行细菌分离鉴定,并对主要病原菌做药敏试验。结果从115份乳样中,分离出6种127株病原菌,其中金黄色葡萄球菌46株,占36.22%,无乳链球菌33株,占25.98%,停乳链球菌21株,占16.53%,大肠埃希菌17株,占13.39%,乳房链球菌8株,占6.3%,沙门菌2株,占1.57%;主要病原菌均对头孢喹诺和左氧氟沙星高度敏感。结论临沂市奶牛乳房炎主要致病菌为金黄色葡萄球菌、链球菌和大肠埃希菌。首选药物为头孢喹诺和左氧氟沙星。因此,治疗奶牛乳房炎应通过药敏试验,合理地选择药物。     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Development of specific and rapid detection of bacterial pathogens in dairy products by PCR

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.     

PCR法快速鉴定眼源性蜡样芽胞杆菌

目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5h内完成,方法灵敏度达7．5～15．0CFU／mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100％,模拟标本的检测灵敏度与纯菌结果一致。结论cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。     

CXCR2基因多态性与奶牛乳房炎和乳品质的关联

采用PCR-SSCP技术研究了荷斯坦牛、西门塔尔牛和通江黄牛3个品种共160头个体CXCR2基因多态性与乳房炎抗性性状和对牛奶品质性状的遗传效应。结果表明: CXCR2基因有3个SNP多态位点, 分别位于序列的第685 bp、777 bp和861 bp位点, 确定了5个等位基因A、B、C、E和F。685 bp位点表现为BC和CC基因型, 777 bp位点表现为AA和AB基因型, 861 bp位点为CC和BC基因型。与奶牛乳房炎敏感性有关的基因型主要是BC、CC和FF, 可能对乳房炎有抗性的是AA、AB和EE基因型。据不同基因型对乳品质的遗传效应分析来看, AA、AB和EE基因型的乳品质性状极显著或显著优于其他基因型。     

Characterization of Staphylococcus aureus strains involved in human and bovine mastitis

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women.     

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.