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。     

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.     

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。     

Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care

After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.     

The usefulness of multiplex PCR for the identification of bacteria in joint infection

Background: The diagnosis of septic arthritis (SA) relies on synovial analysis and conventional culture. But, these methods lack of sensitivity and culture is time consuming to establish a definite diagnosis. This study evaluated a new multiplex PCR assay which entailed screening PCR for Gram typing and identification PCR for species identification using two primer mixes. Methods: A total of 80 synovial fluid samples from patients with suspected SA were collected. Culture, multiplex PCR, and 16S rRNA gene PCR were performed. Results: The analytical sensitivity of multiplex PCR assay was 10¹ CFU/ml for each type of bacteria. There was no cross‐reactivity with common bacterial pathogens. Bacteria were detected in 20, 25, and 26 of 80 samples for culture, multiplex PCR, and 16S rRNA gene PCR, respectively. Nineteen (95%) of 20 culture‐positivesamples and 6 (10%) of 60 culture‐negative samples were positive for the multiplex PCR. Five of six samples which were positive only from multiplex PCR were also positive in 16S rRNA gene PCR. The multiplex PCR showed 2 false‐negative in 27 true‐positive samples but no false‐positive. The sensitivity and specificity of the multiplex PCR were 92.6 and 100%, and the agreement with culture and 16S rRNA gene PCR were 91.3 and 96.3%, respectively. The time to detection for multiplex PCR was a maximum of 6 hr. Conclusion: This multiplex PCR assay offers high sensitivity and improved detection speed relative to culture. The appropriate combination of this new multiplex PCR assay with culture may contribute to the accurate and rapid diagnosis of SA. J. Clin. Lab. Anal. 24:175–181, 2010. © 2010 Wiley‐Liss, Inc.     

16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory.

For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. Sequence analysis of the 16S rRNA gene is a powerful mechanism for identifying new pathogens in patients with suspected bacterial disease, and more recently this technology is being applied in the clinical laboratory for routine identification of bacterial isolates. Several studies have shown that sequence identification is useful for slow-growing, unusual, and fastidious bacteria as well as for bacteria that are poorly differentiated by conventional methods. The technical resources necessary for sequence identification are significant. This method requires reagents and instrumentation for amplification and sequencing, a database of known sequences, and software for sequence editing and database comparison. Commercial reagents are available, and laboratory-developed assays for amplification and sequencing have been reported. Likewise, there are an increasing number of commercial and public databases. Despite the availability of resources, sequence-based identification is still relatively expensive. The cost is significantly reduced only by the introduction of more automated methods. As the cost decreases, this technology is likely to be more widely applied in the clinical setting.     

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.     

重症急性胰腺炎合并细菌感染的快速诊断——附17例检测分析

目的:探讨以16S rRNA基因为基础的细菌PCR法诊断重症急性胰腺炎早期合并细菌感染的价值.方法:17例疑有细菌感染的重症急性胰腺炎病人,术前B超引导下行胰周渗液细针穿刺检查,分别行以16S rRNA基因为基础的细菌PCR法检测和细菌培养.比较细菌PCR法检测结果和细菌培养结果.结果:17例中,PCR检测9例阳性,细菌培养10例阳性,该10例病人行手术治疗,术中所取胰周渗液行细菌培养证实为阳性.PCR检测重症急性胰腺炎合并细菌感染的敏感度为90%,特异度为100%.PCR法和细菌培养法分别需时5 h和3 d.结论:以16S rRNA基因为基础的细菌PCR法能快速、准确地诊断重症急性胰腺炎早期合并细菌感染,为手术治疗提供可靠依据.     

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。     

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.     

聚合酶链反应检测革兰阴性细菌16S rRNA

[目的]寻找诊断细菌感染病原更为敏感和有效的方法.[方法]根据细菌16S rRNA基因的高度保守性,设计合成革兰阴性细菌的共同引物,采用聚合酶链反应检测已知的革兰阴性细菌9株,革兰阳性菌4株.[结果]革兰阴性菌检测阳性率为100%,(检测革兰阳性细菌4株结果均为阴性).采用倍比稀释法检出大肠杆菌的最低浓度为4 CFU/ml.[结论]所用引物具有良好的特异性和敏感性,能达到初步鉴定细菌种属的目的.     

Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

Objectives:  To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.
Methods:  The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.
Results:  The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus .
Conclusions:  A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.     

High-throughput screening of bacterial pathogens in clinical specimens using 16S rDNA qPCR and fragment analysis

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N?=?499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.     

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3% (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.     

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。     

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.     

Real-time PCR for universal antibiotic susceptibility testing

OBJECTIVES: Determination of bacterial antimicrobial susceptibility is usually performed using phenotypic methods. In this study, we developed a universal 16S rRNA and rpoB quantitative PCR assay for susceptibility testing of bacteria commonly isolated in clinical microbiology laboratories. METHODS: Antibiotic susceptibilities for 24 bacterial strains of various species were tested by real-time quantitative PCR assay and by conventional methods. Quantification of DNA copies of either the 16S RNA genes or rpoB were recorded over time in the presence or absence of antibiotics to determine the bacterial growth kinetics and the optimal testing time. RESULTS: Molecular results for antibiotic susceptibility or resistance were in accordance with those obtained using a standard macrodilution broth assay. The method was reproducible, sensitive and rapid (2 h for Gram-negative bacilli and 4 h for Gram-positive cocci). Moreover, this assay was also able to determine the antibiotic susceptibilities of fastidious bacteria, such as mycobacteria, within 5 days. CONCLUSIONS: These results demonstrate that molecular detection of bacteria could be more rapid than phenotypic methods for antibiotic susceptibility testing.     

Combined molecular gram typing and high-resolution melting analysis for rapid identification of a syndromic panel of bacteria responsible for sepsis-associated bloodstream infection

Effective diagnosis and treatment of bloodstream infections are often hampered by a lack of time-critical information from blood cultures. Molecular techniques aimed at the detection of circulating pathogen DNA have the potential to dramatically improve the timeliness of infection diagnosis. Our aim in this study was to establish a rapid, low-cost PCR approach using high-resolution melting analysis to identify a syndromic panel of 21 pathogens responsible for most bloodstream bacterial infections encountered in critical care environments. A broad-range, real-time PCR technique that combines primers for molecular Gram classification and high-resolution melting analysis in a single run was established. The differentiation of bacterial species was achieved using a multiparameter, decision-tree approach that was based on Gram type, grouping according to melting temperature, and sequential comparisons of melting profiles against multiple reference organisms. A preliminary validation study was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. Fifty isolates contained organisms that were present in the panel, and 96% of these were identified correctly at the genus or species level. A correct Gram classification was reported for all 53 isolates. This technique shows promise as a cost-effective tool for the timely identification of bloodstream pathogens, allowing clinicians to make informed decisions on appropriate antibiotic therapies at an earlier stage.     

A case of Bordetella trematum and Kerstersia gyiorum infections in a patient with congestive dermatitis

Bordetella trematum and Kerstersia gyiorum are rare gram-negative bacilli that are not frequently detected in human infections. In this report, we describe a case of a 48-year-old man who presented to our hospital with an infected wound on his leg. Discharges from the cracks of the granulation were collected and evaluated in our microbiology laboratory. Gram staining of the specimen showed polymorphonuclear leukocytes and abundant gram-negative bacilli. Three types of colonies were isolated on blood agar and were identified as B. trematum and Alcaligenes faecalis using VITEK MS. Moreover, K. gyiorum and B. trematum were identified and confirmed via 16S ribosomal RNA (rRNA) gene sequencing. The patient successfully recovered following application of meropenem antibacterial therapy and surgical debridement. This is the first reported case of complex wound infection caused by both B. trematum and K. gyiorum. Identification of B. trematum has recently been made possible by routine bacterial identification using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, K. gyiorum isolation is still rare, and species identification requires 16S rRNA sequencing. Thus, this case highlighted the importance of using multiple methods, such as MALDI-TOF MS and 16S rRNA gene sequencing, for identification of rarely isolated species from clinical specimens.     

Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene

OBJECTIVE: Bacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia. DESIGN: The results of the T-RFLP analysis are compared with two standard biochemical identification methods. METHODS: Lactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed. RESULTS: T-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations. CONCLUSION: T-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.