54株分枝杆菌国际参考株16S rRNA序列分析

目的分析54株分枝杆菌国际参考株16S rRNA序列,为临床分离株的鉴定提供参考。方法用16S rRNA序列分析法对54株分枝杆菌国际参考株进行分析,构建系统发育树,计算相似性百分比。结果除11株(共9种4组)分枝杆菌,即产鼻疽分枝杆菌与塞内加尔分枝杆菌;溃疡分枝杆菌与海分枝杆菌;堪萨斯与胃分枝杆菌;3株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌16S rRNA基因序列完全相同无法相互鉴别,其它41株(共41种)分枝杆菌菌种间16S rRNA均不相同可以得到很好的鉴别。结论 16S rRNA基因序列分析是一种很好的鉴定分枝杆菌的方法,国际参考株16S rRNA基因序列的研究弥补了基因数据库的不足。     

54株分枝杆菌国际标准株16S-23S rRNA转录间隔区序列分析

目的分析54株分枝杆菌国际标准株16S-23SrRNA转录间隔区(ITS)序列,为临床分离株的鉴定提供参考。方法用16S-23SrRNA ITS序列分析法对54株分枝杆菌国际标准株进行分析,构建系统发育树,计算相似性百分比。结果除灰尘与微黄分枝杆菌;田野与千田分枝杆菌;抗热与副偶然分枝杆菌,奥布与母牛结核分枝杆菌;杜氏与猪分枝杆菌;金色与东海分枝杆菌,海与溃疡分枝杆菌、科莫斯分枝杆菌;2株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌的16S-23SrRNA ITS序列完全相同无法鉴别外,其他各分枝杆菌菌种间16S-23SrRNAITS序列均不相同,可以得到很好的鉴别。结论 16S-23SrRNA ITS序列分析是一种很好的鉴定分枝杆菌的方法,国际标准株16S-23SrRNA ITS序列的研究弥补了基因数据库的不足。     

常见分枝杆菌种内不同株之间16S rRNA基因和16S-23SrRNA ITS序列分析结果的比较

目的 针对常见分枝杆菌不同株对其基因序列进行分析,比较分析结果.方法 利用16S rRNA Gene和16S-23S rRNAITS(转录间隔区序列)分析法分别对97株共7种DSMZ/ATCC引进的常见分枝杆菌进行种内不同株之间基因差异性分析,对比两种分型结果的异同.结果 16S rRNA基因可将13株草分枝杆菌分为3...     

不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

目的 评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点.方法 收集41株临床分离的非结核分枝杆菌,以16S rRNA基因测序方法为标准,同时以hsp65基因测序方法及PCR-RFLP方法鉴定菌株,与16S rRNA基因测序结果进行比较.结果 41株非结核分枝杆菌16SrRNA基因测序结果:9株龟分枝杆菌复合群,7株偶发分枝杆菌,7株胞内分枝杆菌,3株鸟分枝杆菌,3株堪萨斯分枝杆菌复合群,3株耻垢分枝杆菌,3株土分枝杆菌,2株草分枝杆菌,2株无色分枝杆菌,1株瘰疬分枝杆菌,1株M.arupense.与16S rRNA基因测序相比较,hs65 PCR-RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌;1株偶发分枝杆菌及1株无色分枝杆菌与其不符;其余菌株鉴定结果一致,符合率为95.1%(39/41).hsp65基因测序结果显示,1株爱尔兰分枝杆菌与16S rRNA测序结果不符,其余菌株鉴定结果与其一致,符合率为97.6%(40/41),并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌.结论 3种方法均能快速鉴定非结核分枝杆菌.与16S rRNA基因测序相比,hsp65基因测序及hsp65 PCR-RFLP更容易鉴定临床最常见非结核分枝杆菌(如堪萨斯分枝杆菌和脓肿分枝杆菌),可在临床推广使用.     

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.     

3株马赛分枝杆菌的分离鉴定及特征分析

摘要:目的：报告对临床分离的3株马赛分枝杆菌的种型鉴定和特征分析。 方法：对3株临床分离株进行生化鉴定、16S rRNA和rpoB基因测序鉴定,用微量肉汤稀释法进行药敏实验。 结果：生化鉴定无法确定种型,且光滑型/粗糙型菌落差异明显;16S rRNA鉴定结果显示,其与龟-脓肿分枝杆菌群高度同源（相似度>99.7%）,联合rpoB基因测序分析可鉴定为马赛分枝杆菌;药敏试验结果显示,其对万古霉素、利奈唑胺、头孢西丁等耐药,其中粗糙型菌耐药性更强。 结论：马赛分枝杆菌是近年来出现的新型致病菌,其粗糙型感染应予以重视。     

16S rRNA基因序列在分析杆菌分类鉴定的研究进展

16S rRNA基因序列高度保守,其核苷酸位点的变化具有种的特异性,用分子生物学技术分析16S rRNA基因,能够将分枝杆菌鉴定到种的水平。本文就用16S rRNA基因进行PCR扩增、DNA直接测序、限制性酶切片段长度多态性（RFLP）分析、DNA探针杂交来鉴定分枝杆菌进行综述。     

3种方法对1株临床疑难菌的鉴定及结果比较

目的 通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)、16S rRNA序列分析和全基因组测序(WGS)对从临床标本中分离的1株疑似盐单胞菌进行菌种鉴定,比较3种方法的鉴定结果。方法 采用MALDI-TOF MS对待测菌进行蛋白质图谱收集,通过比对图谱特征峰实现菌种鉴定;对菌株进行16S rRNA基因测序,将测序结果与数据库比对;待测菌WGS后进行序列比对分析,具体包括使用ANIm和ANIb 2种方法计算平均核苷酸一致性(ANI)以及与基因组分类数据库(GTDB)进行比对;基于全基因组序列中的16S rRNA序列和看家基因序列构建系统进化树,以及基于COG和KEGG功能对基因组进行聚类分析。结果 MALDI-TOF MS对待测菌株的鉴定结果是Halomonas hamiltonii;经16S rRNA序列分析,发现待测菌株与3种盐单胞菌(Halomonas hamiltonii、Halomonas stevensii和Halomonas johnsoniae)的相似度均>99%,且相似度差异非常小,因此无法进行准确的菌种鉴定;基于WGS的基因序列比对、系统发育树、...     

应用基质辅助激光解吸电离飞行时间质谱技术鉴定临床常见厌氧菌

目的：应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术对临床分离的厌氧菌进行鉴定,与传统Vitek32 ANI鉴定卡及16 S rRNA基因序列法鉴定进行比较,分析MALDI-TOF MS技术用于鉴定临床常见厌氧菌的可行性。方法收集从临床标本培养分离的厌氧菌56株,运用 MALDI-TOF MS 技术对其进行鉴定,同时运用 Vitke32 ANI 鉴定卡及16 S rRNA基因序列法分别进行鉴定,将三者的结果进行比较。结果56株厌氧菌中有41株采用 MALDI-TOF MS技术鉴定至种的水平（分值大于或等于2．0）,11株鉴定至属的水平（分值为1．7～2．0）,4株无可靠结果（分值小于1．7）。MALDI-TOF MS和16S rRNA基因序列法鉴定结果一致的占94．6％（53/56）,不一致的占5．6％（3/56）。MALDI-TOF MS与Vitke32 ANI 鉴定卡的鉴定结果一致的占80．4％（45/56）,不一致的占19．6％（11/56）。结论 MALDI-TOF MS与16S rRNA基因序列法鉴定的符合率高,可应用于临床常见厌氧菌的鉴定。     

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。     

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16s rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16s rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.     

基于16S rRNA和gyrB基因的施万菌种水平鉴定分析

  目的   构建基于16S rRNA和gyrB基因对施万菌(Shewanella)进行种水平鉴定的方法,比较2个基因的鉴定能力差异。   方法   利用DnaSP 6.0软件对施万菌16S rRNA 和gyrB 基因的信息位点数及其百分比、核苷酸多态性值、平均G＋C含量、非同义突变率与同义突变率的比值（Ka/Ks）、Tajima检验进行基因多态性分析。 用MEGA 6.06软件的邻接距离矩阵法对90株测试菌株和54株模式菌株构建16S rRNA 和gyrB 基因的进化树。 用MEGA 6.06软件的Kimura’s 2-parameter模型,确定90株测试菌株的菌种后,计算16S rRNA 和gyrB 基因的遗传距离和序列相似性。 比较两者对施万菌种水平鉴定能力差异。   结果   16S rRNA和gyrB基因序列相似性平均值分别为95.0%、80.8%。 在16S rRNA基因进化树中,S. marinintestina和S. sairae、S. livingstonensis和S. vesiculosa的进化分支几乎完全相同,gyrB基因则能在种水平将所有菌株区分开。 16S rRNA基因的种内和种间相似性范围小。   结论   与16S rRNA基因相比,gyrB基因能够更准确的用于施万菌的种水平鉴定。     

Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria

We report an improved method for the detection and identification of mycobacteria using PCR and the heteroduplex mobility shift assay (HMA). The HMA for detection of mycobacteria was based on the microheterogeneity within the DNA coding sequences for 16S rRNA. A remarkable shift between single-stranded, heteroduplex and homoduplex bands in PAGE was observed among the Mycobacterium spp. tested. The Mycobacteria HMA (MHMA) of amplified PCR products from mycobacteria DNA coding for 16S rDNA derived from culture showed a specific heteroduplexes formed among different Mycobacterium species. Other bacterium species were distinguished from Mycobaterium due to slow migrating heteroduplexes mobility bands observed when M. bovis (BCG), M. avium, or M. fortuitum were used as a standard. The specific heteroduplexes were detected when as little as 1 etag of DNA template was used, although better results were obtained with 5 etag and when PCR products of sample test and mycobacterium standard were mixed at a ratio of 1.8. To correctly evaluate the feasibility of using MHMA to detect and identify mycobacteria, 15 clinical sample patients were tested. All MTB-positive clinical samples were identified by MHMA as well as the negative samples. In addition, MHMA will, in principle, be applicable to the detection and classification of any microorganism showing differences within the 16S rRNA as well as to the identification of new and unrecognized bacterial species.     

Molecular identification of nontuberculous mycobacteria isolates in a Brazilian mycobacteria reference laboratory

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.     

Isolation and characterizations of clarithromycin-resistant Mycobacterium avium clinical isolates

Mycobacterium avium is an important intracellular pathogen, particularly in AIDS patients. It also shows the second frequency among nontuberculous mycobacteria infections in Korea. Point mutations of domain V region of the 23S rRNA gene has been known to confer clarithromycin resistance to M. avium. In order to isolate the clarithromycin-resistant strains from clinical isolates of M. avium and characterize them, we isolated the clarithromycin-resistant strains from clinical isolates of M. avium using reverse hybridization assay (RHA) and broth microdilution test (BMT). Three clarithromycin-resistant isolates with high level of MICs were found from 274 clinical isolates by BMT. Two of three resistant strains were also found by RHA, which revealed point mutations in the domain V region of the 23S rRNA. We report here clarithromycin-resistant clinical isolates of M. avium with the different characteristics from those of the resistant strains reported from earlier studies.     

Mycobacterium wolinskyi Infection Confirmed by rpoB Gene Sequencing

Identification of rapidly growing mycobacteria (RGM) is problematic because there are many taxonomic changes. 16S rRNA gene is commonly used to identify Mycobacterium species, but alternative gene targets have been introduced for more accurate identification. We report a rare case of a prosthetic knee infection due to Mycobacterium wolinskyi. The isolate was not identified by 16S rRNA gene sequencing alone and substantially confirmed by rpoB gene sequencing. The identification was delayed because our laboratory did not routinely identify RGM to the species level. Simultaneous sequencing of both 16S rRNA and rpoB genes will allow rapid and accurate identification of M. wolinskyi isolates. J. Clin. Lab. Anal. 26:325‐327, 2012. © 2012 Wiley Periodicals, Inc.     

Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay

The Inno LiPA Mycobacteria assay, based on PCR amplification of the 16-23S rRNA spacer region of Mycobacterium species, has been designed for identification of mycobacteria grown in culture media and discrimination between Mycobacterium tuberculosis complex, M. avium, M. intracellulare, M. kansasii, M. gordonae, M. xenopi, scrofulaceum and M. chelonae group including M. abscessus. In order to evaluate the system as a fast diagnostic tool, the assay was for the first time used directly on 14 microscopy positive clinical specimens and 71 isolates and the results were compared to those of conventional identification using 16S rDNA analysis and biochemical properties. The assay only misidentified one strain, which was found to be M. avium complex instead of M. intracellulare as found by the conventional tests. The assay allows rapid discrimination of the eight most clinically relevant mycobacteria in microscopy positive clinical specimens and isolates within 6 h in the same procedural run.     

A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria

We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.     

耐亚胺培南鲍曼不动杆菌同源性及耐药机制研究

目的调查本院耐亚胺培南鲍曼不动杆菌(IRAB)的分子流行特点,初步研究其对碳青霉烯类抗菌药物耐药的分子机制。方法收集2009年11月至2010年1月不同临床科室分离的IRAB非重复株共54株,用Vitek 2 Compact全自动微生物分析仪进行药敏试验;用脉冲场凝胶电泳(PFGE)分析IRAB的同源性;PCR及DNA测序检测碳青霉烯酶和16S rRNA甲基化酶相关耐药基因型。结果 54株细菌PFGE可分为A、B两个型,其中A型42株,占77.8%,在各科室均有分布,为主要的流行型别;B型10株,占18.5%,仅在神经内、外科的ICU分布。全部54株菌均携带OXA-66基因,53株菌携带OXA-23基因及其上游插入序列ISAba1,41株菌携带16S rRNA甲基化酶armA基因。结论医院临床各科室存在A型IRAB的克隆播散,而B型IRAB仅在神经内、外科ICU流行。OXA-23基因及其上游插入序列ISAba1是最主要的耐碳青霉烯类抗菌药物机制,armA型16S rRNA甲基化酶基因在IRAB中广泛分布。