Detection of Streptococcus anginosus from saliva by real-time polymerase chain reaction

AIMS: The purpose of the present investigation was to assess the salivary levels of Streptococcus anginosus in periodontitis patients. METHODS AND RESULTS: The salivary levels of Strep. anginosus were assessed using real-time polymerase chain reaction (PCR). Streptococcus anginosus was detected in 28 of 37 (75.6%) of periodontitis patients and in three of the 20 (15%) healthy subjects. The mean values for bleeding on probing and probing depth in positive patients were statistically higher than those in negative patients. A significant decrease in Strep. anginosus levels was observed after periodontal treatment. CONCLUSIONS: Although the levels of Strep. anginosus are extremely low, they may reflect the status of periodontal health. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a useful method for obtaining the relative quantities of Strep. anginosus from saliva samples and for monitoring the effect of therapy.     

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

时间分辨荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制整合剂异硫氰酸苯基-EDTA将铕离子标记连接于链霉亲和素分子中,通过光化学反应制备生物素标记pUC118DNA探针,与固定在聚苯乙烯微滴板中的靶DNA杂交后,以铕离子Eu(3+)标记的链霉亲和素为检测物,检测靶DNA的含量,可检测到30pg的靶DNA．     

环介导恒温扩增法检测肺炎链球菌的研究

目的 建立环介导恒温扩增(LAMP)检测肺炎链球菌的方法.方法 用LAMP技术扩增肺炎链球菌菌株,并应用50例临床标本采用传统培养法、PCR法、LAMP法进行检测,比较3种方法的检出率,同时检测方法特异性和灵敏度.结果 所测肺炎链球菌均获扩增产物,对其他非肺炎链球菌无交叉反应.LAMP检测灵敏度可达102 CFU/mL.50例临床标本使用LAMP法检出9例肺炎链球菌阳性(18.0％),使用传统培养法检出阳性4例(8.0％).结论 LAMP法较传统培养检测方法特异性强、灵敏度高、操作方便、快速,适合临床标本的肺炎链球菌检测.     

Dramatic increase in signal by integration of polymerase chain reaction and hybridization on surface of DNA microarray

The cumbersome process required for diagnosis by DNA microarray can be simplified by simple extraction of nucleic acid from cells and by integration of liquid-phase polymerase chain reaction (PCR) and hybridization on the surface of a microarray slide. An unexpected benefit was the large (five- to sixfold) increase in detection signal that also is translated into an increase in sensitivity and the confidence level of diagnosis. The large increase in the detection signal appears to be due to participation of PCR primers as well as to extension of the immobilized capture probes during the hybridization process. The reason for the large increase in signal is not clear in view of only one round of DNA synthesis during the hybridization step. The integrated process correctly identified various genotypes of human papillomavirus (HPV) in the infected clinical human cervical specimens with specificity and efficiency. The process described in this article saves labor, time, and cost and should be applicable for automation of diagnosis by DNA microarray.     

HLA-DR typing using DNA amplification by the polymerase chain reaction and sequential hybridization to sequence-specific oligonucleotide probes

A series of sequence-specific oligonucleotides (SSOs) have been used to type alleles at the HLA-DRB1 locus. Genomic DNA was amplified to high copy number by the polymerase chain reaction (PCR) and hybridizations of the dot-blotted, amplified DNA to a series of 14 SSOs enabled the identification of the major specificities DR1-DRw14. Certain alleles (DR3 and DR4) could be rapidly and accurately identified by running the products of allele-specific amplification of genomic DNA on agarose gels. This approach facilitated the typing of serological specificities such as subtypes of DR3 (DRw17 and DRw18) as well as alleles previously detected by the mixed lymphocyte reaction including subtypes of DR4 (Dw4, Dw10, Dw13, Dw14, and Dw15). The HLA-DR types obtained by SSO probing conformed to rules of Mendelian inheritance when they were applied to a series of 75 families. A full DR type could be obtained from many individuals simultaneously without needing to separate or store viable lymphocytes. Thus, this technique may have considerable implications for the analysis of disease associations with HLA class II alleles, particularly in circumstances where facilities for the initial preparation and storage of the samples may be limited.     

Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization

Harmful algal blooms (HABs) caused by microscopic algae present a threat to human health, ecosystem, fishery, tourism, and aquaculture worldwide. HAB warning and monitoring projects require a simple and rapid method for accurate parallel identification of causative algae. This study presents a useful method for simultaneous detection of harmful algae by multiple PCR coupled with reverse dot blot hybridization (MPCRDBH). A variety of probes, including positive, negative, and specific, were first developed by sequencing and consequent sequence analysis of large subunit rDNA D1–D2 from target species and used for specificity test by blot hybridization. The MPCRDBH assay mainly included five steps: (1) microalgal DNA isolation; (2) amplification and labeling of target DNA by multiple PCR; (3) probe tailing and fixation onto positively charged nylon membrane; (4) reverse dot blot hybridization; and (5) hybridization signal recognition by naked eyes. The reverse dot blot hybridization conditions were optimized, and the appropriate parameters were as follows: ultraviolet cross-linking time, 0.5 min; probe density, 2 μM; Dig-labeled PCR product density, 200 ng; hybridization time and temperature, 2 h and 42 °C; and washing time and temperature, 2 × 5 min and 47 °C. Sensitivity tests showed that MPCRDBH demonstrated a detection limit of 0.6 cell. MPCRDBH recovered all target species and was not affected by background DNA. MPCRDBH also demonstrated a stable detection performance for fixative (acidic Lugol's solution)-preserved samples over 30 d using simulated field samples. MPCRDBH applicability was assessed and proven effective for parallel detection of target microalgae in the field samples. The developed MPCRDBH exhibited a simple membrane-based DNA array preparation and hybridization signal recognition compared with other current DNA arrays. The assay presented in this study is specific and sensitive for parallel detection of microalgae, with stable performance. Therefore, this assay is promising for field monitoring of natural samples.     

Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization

Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP alpha) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group of seven naphthalene-degrading Pseudomonas strains showed strong hybridization with the ndoB probe, and five Gordona, Mycobacterium, Rhodococcus and Pseudomonas strains able to degrade higher molecular weight PAH showed weaker hybridization signals. Using a polymerase chain reaction (PCR) approach, seven naphthalene-degrading Pseudomonas strains showed a PCR fragment of the expected size with ndoB-specific primers and additionally ten strains of Gordona, Mycobacterium, Pseudomonas, Sphingomonas and Xanthomonas able to degrade higher molecular weight PAH were detected with degenerate primer-pools specific for the ISP alpha [2Fe-2S]-Rieske center of diverse aromatic hydrocarbon dioxygenases. This suggests a molecular relationship between genes coding for PAH catabolism in various PAH-degrading bacterial taxa, which could be used to evaluate the PAH-degradation potential of mixed populations.     

建立一种反向斑点杂交法检测肺炎支原体23S rRNAV区A2063G基因突变的方法

目的建立检测肺炎支原体（Mycoplasma pneumoniae,MP）23S rRNA V区2063基因型的反向斑点杂交方法,并与PCR产物直接测序法的结果进行比较分析。方法19例自临床咽拭子标本分离培养的MP,用自行设计的反向斑点杂交法检测23S rRNA V区2063基因型,同时对相应序列测序分析。抽提标准菌株FH和127份经PCR测定MP阳性的临床标本基因组DNA,用MP阴性的基因组DNA抽提物5份作阴性对照,用反向斑点杂交法检测23S rRNA V区2063基因型。结果19例分离培养的MP,经反向斑点杂交法检测15株有23S rRNA V区基因A2063G位点突变;4株为2063A,与测序结果一致（Kappa一致性检验,P〉0.05）。经反向斑点杂交法检测,127份MP阳性的基因组DNA抽提物中有122份标本为A2063G位点突变,标准菌株FH和2份DNA抽提物的2063位点碱基为A,还有3份抽提物标本的2063位点碱基既有A又有G,5份阴性对照均无显色。结论反向斑点杂交方法能快速、准确检测临床MP 23S rRNA V区2063基因型。     

MALDI-TOF MS对肺炎链球菌鉴定与分型的应用

目的 探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法 收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果 除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%（18/60）,分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。     

Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region

Abstract Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non- Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae .     

以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP,将肺炎链球菌基因组DNA的随机酶切片段(200bp～800 bp)克隆到该质粒gfp基因上游的多克隆位点,得到约58000个含有肺炎链球茵基因组DNA随机酶切片段的重组子,提取质粒即为质粒库,该库大约覆盖肺炎链球菌基因组全长的5倍,插入率达到90%以上,且有较强的随机性,质量较高.将该质粒库转化入肺炎链球菌TIGR4菌株,带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后,利用质粒的抗生素抗性基因筛选出重组菌株,从而构建出相应的菌株库,共获得包含约500000个肺炎链球菌转化子的菌株库,经体内、外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告特定条件下的基因表达,并可通过流式细胞仪识别、分选.该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础.     

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time‐correlated single‐photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T‐7 loop of the protein, a region important for the monomer‐monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.     

DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae

The 7-valent polysaccharide conjugate vaccine currently administered against Streptococcus pneumoniae has been shown to be highly effective in high risk-groups, but its use in developing countries will probably not be possible due to high costs. The use of conserved protein antigens using the genetic vaccination strategy is an interesting alternative for the development of a cost-effective vaccine. We have analyzed the potential of DNA vaccines expressing genetically detoxified derivatives of pneumolysin (pneumolysoids) against pneumococcal infections, and compared this with immunization using recombinant protein. The purified recombinant pneumolysoid with the highest residual cytolytic activity was able to confer partial protection against a lethal intraperitoneal challenge, with the induction of high antibody levels. Immunization with DNA vaccines expressing pneumolysoids, on the other hand, induced a significantly lower antibody response and no protection was observed.     

双重荧光定量PCR检测志贺毒素stx1和stx2基因

目的建立一种双重荧光定量PCR检测志贺毒素stx1和stx2基因的方法。方法根据不同细菌来源的stx1和stx2序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度、特异性和重复性评价,并对腹泻患者粪便样本进行检测分析。结果双重实时荧光定量PCR检测含志贺毒素基因重组质粒的最低检测下限为102copies/mL;该法对12种常见肠道病原菌均无特异性扩增,对不同浓度的标准质粒检测重复性高,Ct值变异系数均小于10%;对急性腹泻粪便标本的检测阳性率高于细菌分离培养。结论建立的双重实时荧光定量PCR可作为不同细菌来源的志贺毒素基因的快速鉴定方法,亦可用于人感染性腹泻标本的快速筛查。     

长臂同源多聚酶链反应法构建变形链球菌comD基因同源重组DNA片段

目的构建变形链球菌UAl59密度感应相关的comD基因同源重组DNA片段,为利用同源重组原理构建基因功能丧失菌株做准备。方法通过NCBI基因数据库获取变形链球菌的DNA序列,利用聚合酶链反应技术分别扩增变形链球菌UA159comD基因上、下游片段及抗红霉素基因片段,再通过长臂同源多聚酶链反应将这3个片段连接起来,形成同源重组DNA片段。结果经过PCR反应和琼脂电泳分析,得到了一个碱基数为3个单片段总和的连接片段,测序结果显示连接片段为预期的comD同源重组片段。结论成功构建了变形链球菌UA159comD基因同源重组DNA片段,可直接用于细菌转化构建comD基因缺陷菌株。     

金属蛋白质组学研究锌离子匮乏对肺炎链球菌的影响

【目的】研究锌离子缺乏对肺炎链球菌的影响,找到其适应性生长机制。【方法】以肺炎链球菌为模型,利用加锌和不加锌的培养基对细菌进行培养,收集细胞蛋白,采用双向凝胶电泳,结合金属亲和层析和质谱技术鉴定差异表达蛋白,进而通过生物信息学分析蛋白质相互关系,从中找到细菌适应锌离子匮乏条件的关键代谢通路和蛋白。【结果】测定了在限制培养条件下肺炎链球菌的最适生长浓度,建立了锌离子调控蛋白双向凝胶电泳图谱,鉴定到了96个差异表达蛋白斑点,共67个差异蛋白,其中32个表达下调,35个表达上调,锌离子调控蛋白的作用可能主要体现在糖代谢、核酸代谢、氧化还原作用、辅助蛋白质翻译、合成及折叠等方面。建立了锌结合蛋白的差异表达图谱,鉴定到了10个差异表达蛋白斑点,共7个差异蛋白,其中1个表达下调,6个表达上调。锌离子结合蛋白的作用可能主要体现在应对压力、蛋白质折叠和转运、氨基酸代谢等方面。【结论】肺炎链球菌主要通过调控碳水化合物代谢和核酸代谢等多个代谢通路来应对宿主锌金属离子匮乏的环境,从而使自身能够存活并对宿主形成感染。本研究为揭示细菌在宿主环境,特别是金属离子匮乏条件下的适应性生长机制提供理论基础。     

Detection of Neospora caninum DNA by polymerase chain reaction in bats from Southern China

Neospora caninum is an intracellular protozoan that infects many domestic and wild animals. Domestic dogs and other canids function as definitive hosts, while other mammals serve as natural intermediate hosts. In the present study, the brain tissues of bats collected in Yunnan Province, Southern China were tested by N. caninum specific-nested PCR, targeting the Nc-5 gene and the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA to determine whether bats could be infected with N. caninum. N. caninum DNA was detected in 1.8% (4/227) of bats, i.e., 1.7% (1/60) in Rousettus leschenaultia, 1.7% (1/58) in Hipposideros pomona, 2.9% (2/69) in Rhinolophus pusillus, and none (0/40) in Myotis daubentoniid. The findings of the present study are only the first indication that bats could serve as an intermediate host, and further studies are necessary to confirm whether bats are involved in the transmission of N. caninum infections.