食源性金黄色葡萄球菌脉冲场凝胶电泳分型分析

对石家庄地区食源性金黄色葡萄球菌进行脉冲场凝胶电泳(PFGE)分型,建立石家庄市金黄色葡萄球菌PFGE分型的数据库,探讨引起食物中毒金黄色葡萄球菌菌株和其他食源性金黄色葡萄球菌的分子特征。利用PFGE分型技术对2010至2012年石家庄市的75株食源性金黄色葡萄球菌进行全基因组分析,DNA酶切图谱用BioNumerics软件进行聚类分析。75株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在54%～100%之间,经聚类分析得到45个PFGE型别,6起食物中毒的金黄色葡萄球菌分别由PFGE01型、PFGE02型、PFGE11型、PFGE12型、PFGE15型和PFGE45型引起。建立的金黄色葡萄球菌PFGE分型数据库分子型别较多,且存在差异明显的多个克隆系,对石家庄地区金黄色葡萄球菌引起食物中毒的防控和溯源工作有重要意义。     

金黄色葡萄球菌L型耐药性10年分析

目的 研究金黄色葡萄球菌Ｌ型耐药性变化,确定当前临床用药。方法 对儿科感染性疾病Ｌ型血培养金黄色葡萄球菌进行药敏试验,监测耐药性１０年变化。结果 金黄我葡萄球菌Ｌ型１６９０型,青霉素族,头孢菌素族,大环内酯类,氨基糖甙类及其他抗生素耐药性均升高,丁胺卡那霉素,白霉素,磷霉素,利福平,万古霉素上升幅度较小。结论 金黄色葡萄球菌Ｌ型耐一呈上升趋势,丁胺卡那霉素、白霉素、磷霉素、万古霉素为有效抗生素。     

原位核酸杂交检测宫颈癌中金黄色葡萄球菌L型DNA

应用病原微生物培养、改良革兰氏染色、免疫组化染色及原位核酸杂交等方法,对５０例宫颈癌和３３例慢性宫颈炎进行了研究。结果发现宫颈癌病原微生物培养细菌及其Ｌ型阳性感染率为５８％（２９／５０例）,以金黄色葡萄球菌（金葡菌）及其Ｌ型多见（１３／２９例）;宫颈癌和慢性宫颈炎组织切片革兰氏染色,细菌Ｌ型检出率分别为６６％和４５．５％;免疫组化染色（ＡＢＣ法）金葡菌Ｌ型抗原阳性率各为６８％和４８．５％。Ｌ型主要分布于癌间质、癌巢及宫颈上皮下结缔组织内。１２例宫颈癌金葡菌Ｌ型ＤＮＡ探针原位杂交显示,１０／１２例癌细胞核内有金葡菌Ｌ型ＤＮＡ杂交阳性信号,表明金葡菌Ｌ型ＤＮＡ已进入宫颈癌细胞内,可能会在基因水平上影响宫颈上皮恶变,提示宫颈癌发生与金葡菌Ｌ型感染关系密切,值得进一步研究。     

32株金黄色葡萄球菌分子分型及肠毒素分析

目的对辽宁省内2016-2018年分离出的食源性金黄色葡萄球菌采用脉冲场电泳(PFGE)和肠毒素分型进行分析,为今后公共卫生等领域提供技术保障。方法将32株金黄色葡萄球菌用限制性内切酶SmaI酶切以进行PFGE分析,并用BioNumerics(7.6版本)软件对分离株的指纹图谱进行聚类分析;用PCR方法对菌株进行肠毒素检测。结果 32株食源性金黄色葡萄球菌的PFGE图谱的相似性系数在26.8%~100.0%,经聚类分析得到31个PFGE型别。每株菌均携带1~3种毒力基因,其中每年携带SEH基因最多。结论金黄色葡萄球菌均可以用PFGE和PCR进行分型,都具有较好的分型能力,辨识度高,对菌株有很好的溯源性。     

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。     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

细菌L型败血症324例临床分析

自１９８６年６月至１９９４年２月住院收治细菌Ｌ型败血症３２４例。年龄４天－７８天。病原多为金黄色葡萄球菌。其次为表皮葡萄球菌、大肠杆菌、链球菌、枸椽酸杆菌、鼠伤寒沙门菌、腊样芽胞杆菌、级脓杆菌变形直菌等。做细菌Ｌ型培养同时应用光镜、电镜、酶联免疫法快速诊断,可提高诊断率,使患者早日得到治疗。     

细菌在不同渗透压培养基上的青霉素敏感性

在含ＮａＣｌ０％～１０％的Ｌ型培养基上观察金黄色葡萄球菌、蜡样芽胞杆菌及大肠杆菌对青霉素Ｇ的敏感性和金黄色葡萄球菌Ｌ型对糖的发酵作用和卵磷脂酶活性。结果表明金黄色葡萄球菌、蜡样芽胞杆菌和大肠杆菌在低于、等于或高于细菌细胞内渗透压的培养基上对青霉素的敏感性相似。少数存活的细菌Ｌ型不发酵糖和丧失卵磷脂酶活性,但返祖菌可恢复糖发酵和卵磷脂酶活性。提示细菌细胞内、外渗透压差不是唯一的导致细胞壁缺陷细菌死亡的原因。而胞质周围间隙的破坏,导致其内含酶类的丧失,可能是细胞壁缺陷细菌死亡的重要机制。     

鲫不同种系线粒体DNA物理图谱的构建

用１３种限制性内切酶对鲫鱼三个亚种共七个品系的线粒体ＤＮＡ进行分析,其中有９种酶在种系间或种系内产生限制性片断长度多态性,发现了１６个线粒体ＤＮＡ组合单倍型,通过双酶切分析,构建了１６个线粒体ＤＮＡ组合单倍型的１３种限制性内切酶的发点的物理图谱。     

金葡菌及其L型感染荷瘤小鼠的实验研究

本文应用金黄色葡萄球菌及Ｌ型分别感染肿瘤和非肿瘤５７ＢＬ／６Ｎ小鼠,病理学和细菌学检测发现细菌及Ｌ型感 染种植肿瘤的小鼠组,肿瘤转移率和诱癌能力均显著提高。     

Immunological reagents for comparisons of DNA polymerase-alpha and DNA polymerase-beta

Antibodies to homogeneous calf thymus DNA polymerase-beta and calf thymus DNA polymerase-alpha preparations were raised in rabbits. The antiserum against calf thymus DNA polymerase-beta cross-reacts with all vertebrate DNA polymerase-beta preparations tested, but does not cross-react with trypanosome DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase I. The antibodies against calf thymus DNA polymerase-alpha cross-react with DNA polymerase-alpha from mouse, human, and chicken, but do not cross-react with DNA polymerase-alpha from sea urchin embryos and Drosophila embryos, DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and E. coli DNA polymerase I.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV

The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.     

There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates.

We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication.     

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies.     

Activation of RAW264.7 macrophages by bacterial DNA and lipopolysaccharide increases cell surface DNA binding and internalization

Bacterial DNA containing unmethylated CpG motifs is a pathogen-associated molecular pattern (PAMP) that interacts with host immune cells via a toll-like receptor (TLR) to induce immune responses. DNA binding and internalization into cells is independent of TLR expression, receptor-mediated, and required for cell activation. The objective of this study was to determine whether exposure of immune cells to bacterial DNA affects DNA binding and internalization. Treatment of RAW264.7 cells with CpG oligodeoxynucleotide (ODN) for both 18 and 42 h resulted in a significant increase in DNA binding, whereas non-CpG ODN had no effect on DNA binding. Enhanced DNA binding was non-sequence-specific, inhibited by unlabeled DNA, showed saturation, was consistent with increased cell surface DNA receptors, and resulted in enhanced internalization of DNA. Treatment with Escherichia coli DNA or lipopolysaccharide (LPS) also resulted in a significant increase in DNA binding, but treatment with interleukin-1alpha, tumor necrosis factor-alpha, or phorbol 12-myristate 13-acetate had no effect on DNA binding. Soluble factors produced in response to treatment with CpG ODN or LPS did not affect DNA binding. These studies demonstrate that one consequence of activating the host innate immune response by bacterial infection is enhanced binding and internalization of DNA. During this period of increased DNA internalization, RAW264.7 cells were hypo-responsive to continued stimulation by CpG ODN, as assessed by tumor necrosis factor-alpha activity. We speculate the biological significance of increasing DNA binding and internalization following interaction with bacterial PAMPs may provide a mechanism to limit an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.     

Translocation of nicked but not gapped DNA by the packaging motor of bacteriophage phi29

The biomolecular mechanism that the double-stranded DNA viruses employ to insert and package their genomic DNA into a preformed procapsid is still elusive. To better characterize this process, we investigated packaging of bacteriophage phi29 DNA with structural alterations. phi29 DNA was modified in vitro by nicking at random sites with DNase I, or at specific sites with nicking enzyme N.BbvC IA. Single-strand gaps were created by expanding site-specific nicks with T4 DNA polymerase. Packaging of modified phi29 DNA was studied in a completely defined in vitro system. Nicked DNA was packaged at full genome length and with the same efficiency as untreated DNA. Nicks were not repaired during packaging. Gapped DNA was packaged only as a fragment corresponding to the DNA between the genome terminus and gap. Thus the phi29 DNA packaging machinery tolerated nicks, but stopped at gaps. The packaging motor did not require a nick-free DNA backbone, but the presence of both DNA strands, for uninterrupted packaging.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.