16S rDNA基因芯片检测临床常见感染性细菌

目的 提高细菌和临床微生物检测的速度和准确性，建立含有20种细菌探针在内的感染性细菌检测用基因芯片模型。方法 使用16S rDNA克隆探针和合成的寡核苷酸探针两种，利用点样仪制成基因芯片。细菌DNA经过16S rDNA通用引物扩增后与芯片上的探针杂交，然后用荧光扫描仪检测信号。结果 基因芯片能够用于细菌检测，克服探针具有广泛和灵敏的特点，但是交叉反应明显；寡核苷酸探针具有较高的特异性，但是灵敏度稍差。结论 cDNA探针和寡核苷酸探针结合或设计几个不同的探针来指向同一株细菌很可能是将来基因芯片的检测方向。     

临床常见病原菌16S rDNA单链构象多态性分析

目的原核生物的rRNA基因位点具有高度的保守性,同时不同种属细菌之间又存在相对稳定的变异性,因此可被用于细菌的分类和鉴定。本研究试图利用16SrRNA基因结合分子生物学方法,达到快速鉴定临床常见病原菌的目的。方法收集临床常见各种病原菌333株,筛选2对通用引物在5′端用不同荧光标记,然后对细菌基因组DNA进行聚合酶链反应(PCR)扩增,产物在测序仪上通过毛细管电泳-单链构象多态性(CE-SSCP)分析,通过不同细菌的峰谱不同达到快速鉴定病原菌目的。结果所有受试细菌经过CE-SSCP分析后都有各自不同的峰谱出现,相互之间容易区别。结论利用PCR-CE-SSCP作为病原菌鉴定手段,高效、准确,较传统方法省时,而且灵敏度较高,是一种很有应用前景的细菌快速鉴定方法。     

Comparative study of bacterial flora in bronchoalveolar lavage fluid of pneumonia patients based on their pneumonia subtypes and comorbidities using 16S ribosomal RNA gene analysis

IntroductionThe culture method is the gold standard for identifying pathogenic bacteria in patients with pneumonia but often does not reflect the exact bacterial flora in pulmonary lesions of pneumonia, partly owing to easiness or difficulties in culturing certain bacterial species. We aimed to evaluate bacterial flora in bronchoalveolar lavage fluid (BALF) samples directly obtained from pneumonia lesions using 16S ribosomal RNA (rRNA) gene analysis to compare the results of the BALF culture method in each category of pneumonia.MethodsBacterial florae were detected by a combination of the culture method, and the clone library method using the 16S rRNA gene sequencing in BALF directly obtained from pneumonia lesions in pneumonia patients from April 2010 to March 2020 at the University of Occupational and Environmental Health, Japan, and affiliated hospitals. Clinical information of these patients was also collected, and lung microbiome was evaluated for each pneumonia category.ResultsAmong 294 pneumonia patients (120 with community-acquired pneumonia (CAP), 101 with healthcare-associated pneumonia (HCAP), and 73 with hospital-acquired pneumonia (HAP)), significantly higher percentages of obligate anaerobes were detected in CAP than in HCAP and HAP patients by the clone library method. Corynebacterium species were significantly highly detected in HAP patients and patients with cerebrovascular diseases than in patients without, and Streptococcus pneumoniae was frequently detected in patients with diabetes mellitus.ConclusionObligate anaerobes may be underestimated in patients with CAP. Corynebacterium species should be regarded as the causative bacteria for pneumonia in patients with HAP and cerebrovascular diseases.     

16S rDNA序列在艾伯特埃希菌鉴定中的应用

目的 通过16S rRNA基因序列分析快速鉴定艾伯特埃希菌。方法 以30株疑似艾伯特埃希菌为材料,使用通用引物扩增其16S rRNA基因并进行测序。获得的序列与艾伯特埃希菌型菌株LMG 20976,基因组测序菌株KF1、NBRC107761、TW07627以及其他密切相关的细菌种(大肠埃希菌、赫曼埃希菌、费格森埃希菌、志贺菌和Shimwellia blattae)的16S rRNA基因序列进行比较,并使用N-J法构建进化树来分析其相关性。结果 30株疑似艾伯特埃希菌的16S rRNA基因序列与艾伯特埃希菌参考菌株的序列相似性最高,并与其聚类为一簇,为艾伯特埃希菌。结论 16S rRNA基因测序分析可用于艾伯特埃希菌的快速鉴定。     

16S rDNA实时荧光定量PCR法与全自动培养法在血小板细菌污染检测中的应用比较

目的比较16S rDNA实时荧光定量PCR法与全自动培养法在血小板制品细菌污染检测中的灵敏度和特异性,评价2种方法的应用前景。方法将血小板制品污染中常见的6种细菌用浓缩血小板悬液进行稀释,并选取浓度为102、101、100的菌悬液,分别用实时荧光定量PCR法和全自动培养法进行检测。结果用16S rDNA实时荧光定量PCR法对6株细菌检测,其灵敏度和特异性均为100%,Κ=1.000。用全自动培养仪对6株细菌检测,其特异性均为100%;灵敏度分别为:金黄色葡萄球菌需氧瓶95.5%,Κ=0.886,厌氧瓶90.9%,Κ=0.787;表皮葡萄球菌及大肠杆菌2种培养瓶均为83.3%,Κ=0.667;蜡样芽孢杆菌2种培养瓶均为86.7%,Κ=0.684;铜绿假单胞杆菌需氧瓶度为100%,Κ=1.000,厌氧瓶为44.4%,Κ=0.286;痤疮丙酸杆菌需氧瓶为16.7%,Κ=0.105,厌氧瓶为91.7%,Κ=0.886。结论实时荧光定量PCR法检测血小板制品中的细菌污染,灵敏度高,特异性好,且省时、经济,能应用于临床上血液样本的大规模筛查。     

嗜麦芽寡氧单胞菌临床株与环境株的16S rRNA基因序列及系统发育分析

目的:比较嗜麦芽寡氧单胞菌临床株与环境株16S rRNA基因序列,构建系统发育树,分析其进化关系.方法:对选取的3株嗜麦芽寡养单胞菌临床株和1株环境株的16S rRNA基因进行PCR扩增并测序.将上述及从GenBank中挑选出的其他32株不同来源的嗜麦芽寡养单胞菌的16S rRNA基因序列进行对比分析.并绘制系统发育树.结果:系统发育分析表明大部分菌株可根据来源分为3个簇,序列分析显示某些高度可变区可能存在可区分临床株与环境株的关键序列.结论:嗜麦芽寡养单胞菌基因型及表现型具有多样性:大部分嗜麦芽寡氧单胞菌临床株与环境株可根据16S rRNA基因序列进行鉴别.     

Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood

Universal 16S rRNA gene polymerase chain reaction (PCR) is a promising means of detecting bacteremia. Among other factors, the PCR reagents play a prominent role for obtaining a high sensitivity of detection. The reagents are ideally optimized with respect to the amplifying activity and absence of contaminating DNA. In this study, it was shown in a universal 16S rDNA real-time PCR assay that commercial PCR reagents can vary greatly among each other in these characters. Only 1 of the 5 reagents tested met the criteria of sensitive detection of pathogen DNA with a minimum of false-positive results. The reagent was validated by the detection of pathogens at low titers using bacterial DNA extracted from blood that was spiked with various Gram-positive and Gram-negative bacteria.     

Diagnostic accuracy of a 16S ribosomal DNA gene-based molecular technique (RT-PCR,microarray, and sequencing) for bacterial meningitis,early-onset neonatal sepsis,and spontaneous bacterial peritonitis

The diagnostic accuracy of a 16S ribosomal DNA (rDNA) gene-based molecular technique for bacterial meningitis (BM), early-onset neonatal sepsis (EONS), and spontaneous bacterial peritonitis (SBP) is evaluated. The molecular approach gave better results for BM diagnosis: sensitivity (S) was 90.6% compared to 78.1% for the bacterial culture. Percentages of cases correctly diagnosed (CCD) were 91.7% and 80.6%, respectively. For EONS diagnosis, S was 60.0% for the molecular approach and 70.0% for the bacterial culture; and CCD was 95.2% and 96.4%, respectively. For SPB diagnosis, the molecular approach gave notably poorer results than the bacterial cultures. S and CCD were 48.4% and 56.4% for the molecular approach and 80.6% and 89.1% for bacterial cultures. Nevertheless, bacterial DNA was detected in 53.3% of culture-negative samples. Accuracy of the 16S rDNA PCR approach differs depending on the sample, the microorganisms involved, the expected bacterial load, and the presence of bacterial DNA other than that from the pathogen implied in the infectious disease.     

Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin

Based on 16S rDNA sequence comparison, intracellular mycetome-associated endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) form a distinct lineage within the γ-3 subdivision of proteobacteria, related to the free-living bacterium Escherichia coli , midgut S-endosymbionts of various insects including tsetse flies, and to the P-endosymbiont lineage of aphids, Buchnera aphidicola. Gene organization and expression of several loci in intracellular microorganisms have revealed differences from free-living bacteria. This study analyses two of these characteristics in tsetse endosymbionts; the copy number and gene organization of rDNA operons and the nature of the abundant protein(s) synthesized by these microorganisms. Results indicate that Glossina morsitans morsitans S-endosymbionts have multiple (seven) rDNA operons coding for 16S (rrs) followed by 23S (rrl) gene sequences, whereas tsetse P-endosymbionts have a single, similarly organized rDNA operon. In tsetse mycetocytes in vitro , P-endosymbionts synthesize a predominant protein of 60 kDa in size (p60) which by Western blot analysis shows immunological cross-reactivity with the abundant 63 kDa (p63) protein of B. aphidicola. p63 (also referred to as symbionin) has been characterized as a molecular chaperone, structurally and functionally similar to the groEL protein of E. coli. Under in vitro conditions, tsetse S-endosymbionts synthesize high levels of a similarly-sized protein that cross-reacts with p63 chaperonin. Antisera against the tsetse p60 protein also recognizes p63 protein of B. aphidicola , suggesting that the abundant tsetse endosymbiont protein is a chaperonin.     

16S metagenomics for diagnosis of bloodstream infections: opportunities and pitfalls

Introduction: Bacterial bloodstream infections (BSI) form a large public health threat worldwide. Current routine diagnosis is based on blood culture (BC) but this technique suffers from limited sensitivity. Molecular diagnostic tools have been developed for identification of bacteria in the blood of BSI patients. 16S metagenomics is an open-ended technique that can detect simultaneously all bacteria in a given sample based on PCR amplification of the 16S ribosomal RNA gene (rDNA) followed by sequencing of the PCR amplicons and taxonomic labeling of the sequence reads at genus or species level.

Areas covered: Here we review the studies that have used 16S metagenomics for the identification of bacteria in human blood samples. We also discuss the potential added value of 16S metagenomics in the diagnosis of BSI, challenges as well as future directions for implementation in clinical settings.

Expert commentary: 16S metagenomics has the potential to complement conventional BC; however, the technique currently suffers from several technical limitations jeopardizing implementation in routine clinical microbiology laboratories. Further studies are required to assess the cost-efficiency and clinical impact of 16S metagenomics in comparison to BC which remains the gold standard diagnostic method for BSI.     

Evaluation of 16SpathDB 2.0, an automated 16S rRNA gene sequence database,using 689 complete bacterial genomes

Interpretation of 16S rRNA sequences is a difficult problem faced by clinical microbiologists and technicians. In this study, we evaluated the updated 16SpathDB 2.0 database, using 689 16S rRNA sequences from 689 complete genomes of medically important bacteria. Among these 689 16S rRNA sequences, none was wrongly identified, with 35.8% reported as a single bacterial species having >98% identity with the query sequence (category 1), 63.9% reported as more than 1 bacterial species having >98% identity with the query sequence (category 2), 0.3% reported to the genus level (category 3), and none reported as no match (category 4). For the 16S rRNA sequences of non-duplicated bacterial species reported as category 1 or 2, the percentage of bacterial species reported as category 1 was significantly higher for anaerobic Gram-positive/Gram-negative bacteria than aerobic/facultative anaerobic Gram-positive/Gram-negative bacteria. 16SpathDB 2.0 is a user-friendly and accurate database for 16S rRNA sequence interpretation in clinical laboratories.     

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。     

血液细菌16S rRNA基因检测的诊断价值

目的探索快速可靠的检测细菌感染的新方法。方法用PCR技术扩增实验室保留株10株的16SrRNA基因,以乙型肝炎病毒-DNA、白假丝酵母菌和人类基因组DNA为对照,检测该方法的特异性;采用10倍比稀释法进行该方法的灵敏度检测。结果对所测细菌株均获得475bp扩增产物,而与乙型肝炎病毒-DNA、白假丝酵母菌和人基因组DNA无交叉反应;PCR最低能检测1.5×104/L大肠埃希氏菌。结论16SrRN基因PCR检测细菌感染的方法具有特异性、快速性和敏感性高等特点。     

用16S rRNA基因克隆文库研究腹泻粪便中菌群分布

目的 建立16S rRNA基因克隆文库分析菌群的方法,用于临床标本菌群分布的检测.方法 临床标本直接提取核酸,用16S rRNA基因的通用引物进行PCR扩增、纯化、连接、克隆和测序,建立16s rRNA基因克隆文库,序列与数据库进行比对分析.结果 通过16S rRNA基因克隆文库分析,腹泻标本中检出4种细菌,脆弱拟杆菌为绝对优势菌,占91%;腹泻恢复后的粪便标本中菌群呈多样性,检出12种确定种属的细菌,其中脆弱拟杆菌占14%.结论 16S rRNA基因克隆文库是一种较好地研究粪便标本菌群的方法,可直接进行粪便标本的菌群分析和研究各种细菌在腹泻中的作用.     

海南省类鼻疽临床分离株核糖体16S-23S内转录间隔区序列多态性分析

目的 研究海南省类鼻疽伯克霍尔德菌核糖体16S-23S内转录间隔区（ITS）序列多态性及基因型特征,了解该省与其他类鼻疽流行区菌株间的遗传背景差异。方法 PCR扩增272株类鼻疽伯克菌的ITS片段并通过毛细管凝胶电泳检测产物长度;通过DNA测序及序列比对确认不同长度ITS的基因型及序列一致性;通过2检验分析不同流行区ITS型别的分布差异。结果 经毛细管凝胶电泳及DNA测序确认,272株类鼻疽伯克菌中发现C、E、CE、G 4种ITS基因型:C型64株（23.53%）,E型144株（52.94%）,CE型56株（20.59%）,G型8株（2.94%）。序列比对证实,海南省类鼻疽伯克菌中各型别（C、E、G）ITS序列高度保守,不同型别ITS的长度变化由其主要变异区的序列差异引起。我国海南省与泰国流行区的ITS型别分布差异有统计学意义（2=8.296,P0.05）,与澳大利亚流行区分布差异无统计学意义（2=5.521,P0.05）。结论 海南省类鼻疽伯克菌临床菌株中存在C、E、CE、G 4种ITS基因型,C、E、CE为优势型别;与泰国、澳大利亚等传统流行区相比,其G型菌株比例较高。     

应用16S rRNA宽范围聚合酶链反应快速检测临床无菌体液感染

目的应用16S rRNA宽范围聚合酶链反应（PCR）快速检测临床无菌体液感染,并与传统培养方法进行比较分析。方法收集94份临床无菌体液（血液、脑脊液、胸腹水、关节液）标本,提取细菌基因组DNA,应用16S rRNA宽范围PCR扩增,并将PCR阳性产物测序,然后将测序结果在美国国家生物技术信息中心（NCBI）上进行BLAST比对,从而确定菌种。同时,应用常规培养方法检测94份无菌体液标本,并对2种方法的结果进行比较。结果 16S rRNA宽范围PCR敏感性高,最低扩增浓度为103 CFU/mL,且该技术特异性高,其阳性扩增产物经测序比对后结果和培养结果完全吻合。另外,94例临床标本中应用培养方法培养出阳性例数为9例,阳性率为9.6%,而应用宽范围PCR,除培养阳性的9例均为阳性外,另外扩增出6例阳性标本,阳性率为15.9%,而此6例经测序证实分别为4株铜绿假单胞菌、1株黏质沙雷菌、1株肺炎链球菌。结论 16S rRNA宽范围PCR与培养技术比较起来,敏感性高,特异性强,有望成为临床无菌体液病原体感染快速筛查的方法。     

30株苍白杆菌的分离鉴定和药物敏感性分析

目的分析临床苍白杆菌分离株的种型特征和药物敏感性。方法收集临床分离苍白杆菌共30株。用手工生化试验、Vitek 2 Compact GN鉴定卡、API 20NE鉴定试条进行生化鉴定,扩增16S rRNA基因并进行测序鉴定和序列分析。用Vitek 2Compact AST-GN13药敏鉴定卡进行微量稀释法的药敏实验。结果经生化鉴定和基因测序,17株为人苍白杆菌、10株为中间苍白杆菌、1株为嗜血苍白杆菌、1株疑为苍白杆菌属内未分类的新种、1株为解糖精假苍白杆菌。药敏试验显示对喹诺酮类和亚胺培南的敏感率分别为100%和93.3%;对三代头孢类抗菌药物和哌拉西林/他唑巴坦的耐药率为93.3%和96.7%;对氨基糖苷类抗菌药物敏感性因菌种而异,人苍白杆菌对阿米卡星、庆大霉素和妥布霉素的敏感率分别为94.1%、88.2%和94.1%,而中间苍白杆菌为0%、10%和0%,差异具有统计学意义(χ2=14.50,P0.05)。结论临床苍白杆菌感染主要由人苍白杆菌和中间苍白杆菌引起,二者对氨基糖苷类抗菌药物敏感率的差异可用于其种型的区分。     

质粒介导16S rRNA甲基化酶肺炎克雷伯菌临床分离株的检测及分子流行病学研究

目的 调查165 rRNA甲基化酶在肺炎克雷伯菌临床分离株中的流行情况.方法 收集我院2006年1月至2007年9月从临床标本中分离的肺炎克雷伯菌337株,所有菌株均为非重复株.菌种鉴定采用全自动微生物分析仪,庆大霉素、阿米卡星和妥布霉素的药敏试验采用琼脂稀释法.超广谱B内酰胺酶(ESBLs)检测采用美国临床和实验窒标准协会(CLSI)规定的纸片确证法.使用PCR检测16S rRNA甲基化酶基因、整合酶基因和ESBL基因.接合试验检测质粒的可转移性;脉冲场电泳分析菌株的同源性.结果 337株肺炎克雷伯菌对庆大霉素、妥布霉素和阿米卡星的耐药率分别为19.0%(64/337)、8.3%(28/337)和16.3%(55/337).21株16S rRNA甲基化酶基因阳性,阳性率为6.2%(21/337),其中13株rmtB阳性、3株armA阳性、5株rmtB和atmA同时阳性.所有16S rRNA甲基化酶基因阳性株对庆大霉素、妥布霉素和阿米卡星同时耐药且都为高度耐药(MICs≥256μL),21株甲基化酶基因阳性株有19株产ESBLs,ESBL基因主要为CTX-M-14-like、CTX-M-15-like和SHV-12-like.21株均为Ⅰ类整合酶基因阳性.13株通过接合试验把质粒传递给受体菌E. coliJ53.所有接合子Ⅰ类整合酶基因阳性、blaTEM-1基因阳性、产ESBLs及对庆大霉素、妥布霉素、阿米卡星和复方磺胺甲噁唑耐药,对其他抗菌药物敏感.接合子ESBL基因型与供菌一致.21株16S rRNA甲基化酶基因阳性株经脉冲场凝胶电泳(PFGE)分成14个基因型,主要为A型和Ⅰ型.结论 armA和rmtB型16S rRNA甲基化酶基因已经在肺炎克雷伯菌中播散,既可通过克隆株播散也可通过接合性质粒在不同菌株间播散.