痰标本中金黄色葡萄球菌的分离鉴定方法研究

目的：探索一种基于16S rRNA 基因的细菌快速鉴定方法,为临床标本中未知病原菌的诊断提供新的方法思路。方法对1份临床发热伴呼吸道症候群患者的痰标本进行分离培养,获得3种不同的菌落。直接以纯菌落为模板,以通用引物扩增未知菌的16S rRNA 基因片段,产物直接测序后进行基于局部比对算法的搜索工具比对,根据序列同源性鉴定病原细菌。结果3种菌落分别为不易致病的表皮葡萄球菌、龋齿罗特放线菌及致病性的金黄色葡萄球菌,后者继续用特异性引物扩增检测鉴定,确认为金黄色葡萄球菌。结论本研究建立了一种利用16S rRNA 基因扩增快速鉴定临床标本中未知病原菌的简便方法。     

细菌性肺炎快速诊断基因芯片的初步构建

目的：根据细菌16 S rRNA基因特点设计常见病原菌的特异性探针,采用酶显色技术构建基因芯片,探讨其临床应用的可能性。方法选取肺炎链球菌、流感嗜血杆菌及铜绿假单胞菌等8种细菌性肺炎常见的病原菌的标准菌株作为研究对象,并选择12份患者的痰液标本进行检测。在16 S rRNA基因保守区设计 PCR反应的通用引物及革兰阳性菌、革兰阴性菌的通用探针,利用可变区的差异设计合成特异性探针,构建基因芯片。利用细菌16S rRNA基因设计的PCR通用引物进行扩增,所有8种细菌均获得350 bp的扩增产物。以地高辛标记特异性探针,构建完成可用于8种常见病原菌检测的基因芯片,结果8种标准菌株基因芯片检测均取得了预期效果,对12份痰标本中常规培养阳性7份,其对应芯片检测结果均成阳性,5份常规培养阴性的标本中,芯片结果提示阳性的有3份,其中1份为嗜肺军团菌,2份为使用抗生素后的患者标本。结论设计合成的 PCR通用引物对扩增细菌的16 S rRNA基因具有较高的特异性及灵敏度。构建的基因芯片可用于常见细菌性肺炎病原菌的检测鉴定,且对抗生素使用后的临床标本及苛养菌有一定的诊断价值。本研究所获得基因芯片对于细菌性肺炎的检测具有简单、快速、特异性及敏感性高的特点。     

16SrRNA、16S-23SrRNA基因测序分析检测主要血流感染病原菌比较

目的比较细菌16SrRNA、16S-23SrRNA基因测序分析在血流感染病原菌检测中的作用。方法提取临床上血流感染常见的金黄色葡萄菌、表皮葡萄球菌、大肠埃希菌、粪肠球菌、肺炎链球菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、洛菲不动杆菌、肺炎克雷伯杆菌、化脓性链球菌、奇异变形杆菌、潘尼变形杆菌、屎肠球菌、粘质沙雷菌、宋内志贺菌、产气肠杆菌、小肠结肠炎耶尔森菌、腐生葡萄球菌基因组DNA,运用16SrRNA、16S-23SrRNA基因进行PCR扩增。扩增产物经测序后在美国国家生物技术中心（NCBI）上进行比对分析,确定菌种。结果在所分析的19种临床血流感染常见细菌中,16SrRNA基因测序分析可将除粘质沙雷菌外的细菌鉴定到种的水平,但无法完全区分近缘种属;16S-23SrRNA成功鉴定17种细菌,除大肠埃希菌、宋内志贺菌外所有细菌均成功鉴定到单一种的水平。结论16S-23SrRNA基因可作为血流感染细菌检测较好的分子靶标。     

16S rRNA 测序鉴定1株条件致病性人苍白杆菌

目的：探索一种基于16S rRNA 基因的细菌快速鉴定方法,为临床未知病原菌的诊断及治疗提供科学依据。方法对临床患者的痰标本分离培养纯菌落,直接以菌液为模板,以通用引物 PCR 扩增未知菌的16S rRNA 基因片段,产物直接测序。将测序结果进行 BLAST 比对,根据序列同源性鉴定病原细菌。结果未知病原菌经本实验鉴定为人苍白杆菌,经 ABI 细菌快速鉴定板条检测,确认结果一致。结论该研究简化了临床标本未知病原菌分离培养鉴定的步骤,建立了一种利用16S rRNA 基因扩增快速鉴定病原菌的简便方法。     

病原生物学检验与临床

导尿管伴随性尿路感染细菌学检测的样本采集和处理；两步聚合酶链反应法快速诊断感染性角膜炎和眼内炎病原体的研究；新生儿呼吸机相关肺炎病原菌分析；105株新生儿败血症血培养病原菌及药敏结果分析；23S rRNA基因在常见病原菌鉴定中的应用；Phoenix-100全自动微生物鉴定／药敏系统检测耐甲氧西林葡萄球菌；肠球菌氨基糖苷类高水平耐药基因的检测。     

多重PCR同时检测病原菌及其耐药基因

目的建立一种多重PCR方法,在同一扩增体系内同时检测病原菌及其β-内酰胺类耐药基因,探讨采用多重PCR同时鉴定病原菌及其耐药基因的可行性。方法根据细菌基因序列和对β-内酰胺类抗生素耐药特点,设计一对细菌鉴定通用引物和三对β-内酰胺类耐药基因引物,并在同一扩增体系内行PCR扩增。结果 MRSA和大肠埃希菌及肺炎克雷伯菌的产酶标准菌株的bla mecA、blaTEM、blaSHV和16S-23S rRNA基因间隔区（ISR）多重扩增均为阳性,对本实验室内保存的50株多重耐药的葡萄球菌（包括40株表型筛查为MRS菌株及10株非MRS菌株）及30株ESBLs阳性的大肠埃希菌及30株肺炎克雷伯菌的多重PCR结果显示：MRS阳性菌株均同时扩增出了葡萄球菌特有的多态性及blamecA指纹图谱,而多重耐药的非MRS菌株也都扩增出了blamecA,ESBLs表型阳性的大肠埃希菌多以blaTEM为主,而肺炎克雷伯菌多以blaSHV为主。结论多重PCR与传统的培养及药敏试验相比敏感、特异、迅速,对于解决难培养或不能培养的微生物的鉴定和药敏试验,是一种很有前景的方法。     

23S rRNA基因在临床常见致病菌检测中的应用

目的 根据临床常见致病菌23S rRNA基因序列的差异，建立可初步鉴别革兰阴性菌和革兰阳性菌的分子生物学方法。方法 分析常见细菌的23S rRNA基因序列，设计相应引物。采用多重PCR扩增标准菌株及临床分离株23S rRNA基因，并根据电泳结果作出初步分类。结果 革兰阴性菌株均出现1条DNA电泳条带(约为350bp)，而革兰阳性菌株则出现2条电泳条带(约为250和400bp)。60株临床分离菌经PCR扩增、电泳后，电泳分析结果与常规鉴定结果符合率达100％。结论 23S rRNA基因检测用于细菌初步分类鉴定，具有快速、灵敏、准确的特点，可为细菌感染的实验室诊断提供客观依据。     

荧光定量PCR/熔解曲线分析法快速鉴定葡萄球菌菌种

目的：建立一种基于葡萄球菌16～23s rRNA间隔区序列特征的荧光定量PCR／熔解曲线分析鉴定方法，以快速鉴定葡萄球菌菌种。方法：在葡萄球菌16s rRNA 3′端和23s rRNA5′端分别设计上、下游引物．用荧光定量PCR方法扩增各个实验菌株的16～23s rRNA间隔区序列。然后分析各扩增产物的熔解曲线．以确定各个菌种熔解曲线特征，并以此为依据对15株葡萄球菌临床分离株进行鉴定。结果：设计的引物对成功地扩增了葡萄球菌所有实验菌株，对扩增产物的熔解曲线分析可初步鉴定金黄色葡萄球菌、表皮葡萄球菌和溶血葡萄球菌。以VITEK2鉴定结果为标准，荧光定量PCR／熔解曲线分析法鉴定菌种的准确率为67％（10／15）．整个实验可在1h内完成。结论：荧光定量PCR／熔解曲线分析法有望成为临床实验室快速鉴定葡萄球菌菌种的新方法。[著者文摘]     

16S rRNA通用引物在血小板制品细菌污染检测中的应用

目的评价16S rRNA通用引物在快速检出血小板制品中污染细菌的实用性,寻找新的能够快速、准确地检出血小板中污染细菌的方法。方法用分子生物学方法提取样本中细菌DNA,用设计合成的16S rRNA和16S—23S rRNA基因区间引物进行扩增,产物经HaeⅢ酶切,酶切片段与血小板污染常见菌的酶切图谱对比,确定有无污染及污染菌种类。结果16S rRNA通用引物法可以在4h内完成全部检测,其中在保存24h的血小板标本中未检出污染菌,保存48h的血小板标本中有2份分别检出了金黄色葡萄球菌和枯草芽孢杆菌,与全自动细菌培养仪的结果一致。结论16S rRNA通用引物是一种快速、准确检出血小板制品中污染细菌的方法,结果可靠,具有较高的临床应用价值。     

应用基因芯片快速检测临床常见细菌

目的 应用基因芯片对临床常见的8种致病细菌进行检测.方法 选取8种临床常见的致病细菌,包括金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、大肠杆菌、奇异变形杆菌、产气肠杆菌、荧光假单胞菌、宋内志贺菌.以16S rRNA基因为目的基因,白行设计通用引物系列扩增目的片段,针对高变区域设计探针,建立基因芯片检测体系,并对所选细菌...     

一种快速同步检测肺炎支原体及其大环内酯类耐药突变的新方法

目的 建立基于CycleavePCR技术的肺炎支原体及其大环内酯类耐药突变株快速检测方法.方法 收集102株分离自2005-2008年上海市交通大学附属儿童医院住院患儿的肺炎支原体及136份2009年11-12月上海市交通大学附属儿童医院呼吸道感染的住院患儿经鼻采集鼻咽部痰标本,根据肺炎支原体无突变敏感株与突变耐药株23S rRNA基因2063/2064位的碱基差异及上下游保守序列分别设计新型特异性探针(Cycling探针)及引物,建立肺炎支原体及其大环内酯类耐药突变株检测方法,以含有被检测靶序列的重组质粒为阳性对照,以常见呼吸道病原菌DNA为阴性对照,评价其敏感度及特异度.采用自建方法检测所有标本,并与常规PCR及测序结果比较.结果 该方法可准确检出并鉴别大环内酯类敏感及耐药突变株阳性对照,102株临床分离株中83株对大环内酯类耐药,测序表明82株发生23S rRNA基因A2063G突变,1株A2064G突变,与CycleavePCR检测结果相符,136份痰标本检测结果也与常规PCR扩增及测序结果相符.所有阴性对照样品均未出现假阳性.与常规PCR及测序方法相比,CycleavePCR法的敏感度和特异度均达100%.该方法的检测下限为10拷贝/PCR反应,扩增检测可在1.5 h内完成.结论 建立了一种基于CycleavePCR技术的肺炎支原体及其大环内酯类耐药突变株快速检测方法,可同步提供病原菌及其耐药性信息,为肺炎支原体感染诊断及临床合理选用抗菌药物提供参考.     

Comparison between two PCR-based bacterial identification methods through artificial neural network data analysis

The 16S ribosomal ribonucleic acid (rRNA) and 16S-23S rRNA spacer region genes are commonly used as taxonomic and phylogenetic tools. In this study, two pairs of fluorescent-labeled primers for 16S rRNA genes and one pair of primers for 16S-23S rRNA spacer region genes were selected to amplify target sequences of 317 isolates from positive blood cultures. The polymerase chain reaction (PCR) products of both were then subjected to restriction fragment length polymorphism (RFLP) analysis by capillary electrophoresis after incomplete digestion by Hae III. For products of 16S rRNA genes, single-strand conformation polymorphism (SSCP) analysis was also performed directly. When the data were processed by artificial neural network (ANN), the accuracy of prediction based on 16S-23S rRNA spacer region gene RFLP data was much higher than that of prediction based on 16S rRNA gene SSCP analysis data (98.0% vs. 79.6%). This study proved that the utilization of ANN as a pattern recognition method was a valuable strategy to simplify bacterial identification when relatively complex data were encountered.     

DNA probes for Bordetella species and a colorimetric reverse hybridization assay for the detection of Bordetella pertussis.

Three oligonucleotide probe sequences were inferred from the 16S ribosomal ribonucleic acid (rRNA) and the 16S-23S rRNA spacer sequences of Bordetella pertussis ATCC 10380. These probes were used in hybridization tests with deoxyribonucleic acid from Bordetella species and other relevant bacterial taxa. A probe from the spacer region hybridized exclusively to the B. pertussis strains tested and not to strains from other species. Using a combination of three probes, B. pertussis, B. parapertussis/B. bronchiseptica and B. avium could be specifically identified and differentiated from other taxa. Differentiation between B. parapertussis and B. bronchiseptica was not possible with the probes used. Using the spacer probe, a colorimetric hybridization assay specific for B. pertussis was developed based on enzymatic amplification of the 16S-23S rRNA spacer and reverse hybridization in microtitre wells. As compared with results using agarose gel electrophoresis, and Southern and dot-spot hybridization with a 32P-labelled probe, this assay proved to be faster and easier to perform and was found to be at least as sensitive and specific.     

Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples

A membrane-array method was developed for the detection of human intestinal bacteria in fecal samples without using the expensive microarray-arrayer and laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or pure cultured bacteria using two universal primers, and were hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP color development. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens, Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the membrane-array method is a reliable technique for the detection of predominant human intestinal bacteria in the fecal samples. The result was also confirmed by using specific PCR methods for these bacteria.     

肠球菌菌种鉴定方法的探索

目的 选择一种能将肠球菌属鉴定到种水平的快速准确的方法.方法 分别使用生化鉴定方法、16SrRNA基因测序技术、rpoA基因测序技术、基因组杂交技术(DNA-DNA hybridization,DDH)和平均核苷酸相似度(Average nucleotide identity,ANI)对12株肠球菌进行种水平鉴定,并比...     

反向斑点杂交快速检测肺炎链球菌

目的:建立一种利用DNA探针快速检测肺炎链球菌的反向斑点杂交方法.方法:在肺炎链球菌特异的自溶素基因序列内设计引物,聚合酶链反应合成1段263 bp的DNA探针,然后用生物素标记的细菌DNA与该探针行反向斑点杂交,并将该方法应用于痰样本的检测.结果:所合成的探针具有高度特异性,与其他细菌间无交叉反应,该方法能检测出10 ng细菌DNA.50份痰标本肺炎链球菌培养阳性者8份,杂交阳性者12份,PCR阳性者18份.结论:反向斑点杂交具有快速、敏感、特异的优点,对肺炎链球菌的快速诊断有重要意义.     

DNA microarray analysis of predominant human intestinal bacteria in fecal samples

A microarray method was developed for the detection of 40 bacterial species reported in the literature to be predominant in the human gastrointestinal tract. The 40 species include seven species each of Bacteroides and Clostridium, six species of Ruminococcus, five species of Bifidobacterium, four species of Eubacterium, two species each of Fusobacterium, Lactobacillus and Enterococcus, and single species each of Collinsella, Eggerthella, Escherichia, Faecalibacterium and Finegoldia. Three 40-mer oligos specific for each bacterial species were designed based on comparison of the 16S rDNA sequences available in the GenBank database, and were used to make the DNA-array on epoxy slides. Using two universal primers, the 16S rRNA gene from bacteria present in fecal samples were amplified and labeled with Cyanine5-dCTP by PCR, and then hybridized to the DNA-array. After resolving some difficulties caused by sequence conflicts in GenBank and inaccurate reference strains, all 40 bacterial reference species gave positive results. The microarray method was used to screen fecal samples obtained from 11 healthy human volunteers for the presence of these intestinal bacteria. The results indicated that 25-37 of the 40 species could be detected in each fecal sample and that 33 of the species were found in a majority of the samples.