16S rRNA基因PCR在新生儿败血症诊断研究中的应用

１６ＳｒＲＮＡ基因ＰＣＲ在新生儿败血症诊断研究中的应用滕懿群孙眉月尚世强洪文澜朱方远于钟声参考有关文献自行设计、经计算机辅助同源性分析，以细菌共有的细菌核糖体１６ＳＲＮＡ（１６ＳｒＲＮＡ）基因高度保守区为引物，对引起新生儿败血症常见的金黄色葡萄球菌、...     

对Rett综合征中发现的rRNA基因突变进行计算机辅助的二级 …

目的 分析Ｒｅｔｔ综合征中ｒＲＮＡ基因突变ｍｔＤＮＡ２８３５＾Ｃ→Ｔ和ｍｔＤＮＡ２７０６＾Ａ→Ｇ引起的ｒＲＮＡ二级结构变化。方法 将ｒＲＮＡ基因的野生型和突变后的ｍｔＤＮＡ序列共同输入计算机,以软件ＤＮＡＳＩＳ（６．１４版）加以分析。统一设置模拟长度为３００碱基,最大环径３０碱基。结果 ｍｔＲＮＡ２８３５＾Ｃ→Ｔ与阳性对照一样,使１６ＳｒＲＮＡ二级结构彻底变化,能量变化很大;相反,ｍｔＤＮＡ２７０     

荧光原位杂交法检测双歧杆菌

细菌的分类鉴定按常规需通过细菌形态观察、菌壁成份分析和发酵产物的鉴定 ,以及遗传特征如ＤＮＡ的Ｇ Ｃ百分含量和ＤＮＡ的同源性分析来完成。这些方法对于某些细菌尤其是那些目前尚无法培养的细菌鉴定并不理想。自从Ｄｅｌｏｎｇ等首次采用荧光素标记的寡核苷酸基因探针成功地对单个完整细胞进行分类鉴定以来 ,国外许多研究者对这种新方法作了各种尝试 ,从纯培养细菌到混合物中的细菌 ,原位荧光杂交法都获得了令人满意的效果。细胞内核糖体数量达 10 4 ～ 10 5,核糖体ＲＮＡ(ｒＲＮＡ)又具有特异区和保守区之分 ,因此 5ＳｒＲＮＡ和 16…     

阴道加德纳菌ITS-23S rRNA部分基因克隆及序列分析

目的 分析深圳地区阴道国纳菌（Ｇ．ｖａｇ）ＩＴＳ（ｉｎｔｅｒｎａｌ ｔｒａｎｓｃｒｉｂｅｄ ｓｐａｃｅｒ）－２３Ｓ ｒＲＮＡ部分基因序列。方法 设计特异性引物,建立检测Ｇ．ｖａｇ的ＰＣＲ方法,对临床病人阴道分泌物标本进行检测,并对分离出的４株ＰＣＲ产物进行分子克隆和序列分析。结果 分离出的４株Ｇ．ｖａｇＩＴＳ－２３ｓ ｒＲＡＮ基因的核苷酸同源性的９９．５％以上,与Ｂｅｌｄｕｍ报道株同源性在８９％以     

多重半套式聚合酶链反应检测脑脊液中细菌16S rRNA基因

根据细菌 16Ｓ核糖体ＲＮＡ(ｒｉｂｏｓｏｍａｌＲＮＡ ,ｒＲＮＡ)基因兼有保守性和变异性 ,以及对于本应无菌的体液标本 (如血液、脑脊液 ) ,只要确定有细菌存在即可断定为感染之特点 ,建立了多重半套式聚合酶链扩增 (ｍｕｌｔｉｐｌｅｘｓｅｍｉ ｎｅｓｔｅｄＰＣＲ)的方法 ,对脑脊液标本中细菌的感染进行检测。在ＧＥＮＥＢＡＮＫ中查得常见细菌 16ＳｒＲＮＡ基因序列 ,用ＤＮＡｓｔａｒ软件进行分析 ,在保守区Ｕ3 和Ｕ8各寻找一段序列作为细菌的通用引物 (上游引物Ｐｕ3:5′ＴＡＣＧＴＧＣＣＡＧＣＡＧＣＣＧＣＧＧＴＡＡＴＡ3′ ,下游…     

中国莱姆病螺旋体rRNAA基因多态性分析

本文作者首次应用地高辛标记的大肠杆菌１６＋２３ＳｒＲＮＡ基因探针分析中国莱姆病螺旋体ｒＲＮＡ基因限制性图谱。４５株中国菌株和６株国外菌株用于实验，选用ＥｃｏＲⅤ和ＨｉｎｄⅢ两种限制性内切酶。实验结果表明，中国菌株遗传差异性较大，分属三个基因种，即Ｇｅｎｏｓｐｅｃｉｅｓ Ⅰ，ＧｅｎｏｓｐｅｃｉｅｓⅡ和ＧｅｎｏｓｐｅｃｉｅｓⅢ，其中Ｂ．ｂｕｒｇｄｏｒ ｆｅｒｉ Ｓｅｎｓｕ Ｓｔｒｉｃｔｏ基因种在亚洲菌     

对Rett综合征中发现的rRNA基因突变进行计算机辅助的二级结构模拟分析

目的分析Ｒｅｔ综合征中ｒＲＮＡ基因突变ｍｔＤＮＡ２８３５Ｃ→Ｔ和ｍｔＤＮＡ２７０６Ａ→Ｇ引起的ｒＲＮＡ二级结构变化。方法将ｒＲＮＡ基因的野生型和突变后的ｍｔＤＮＡ序列共同输入计算机,以软件ＤＮＡＳＩＳ（６．１４版）加以分析。统一设置模拟长度为３００碱基,最大环径３０碱基。结果ｍｔＤＮＡ２８３５Ｃ→Ｔ与阳性对照一样,使１６ＳｒＲＮＡ二级结构彻底变化,能量变化很大;相反,ｍｔＤＮＡ２７０６Ａ→Ｇ引起的结构和能量变化都很小。结论计算机模拟程序能对这两种突变引起的ｒＲＮＡ结构变化进行快速的预测,其中ｍｔＤＮＡ２８３５Ｃ→Ｔ突变可能与Ｒｅｔ综合征发病有关,而另一个突变ｍｔＤＮＡ２７０６Ａ→Ｇ则对二级结构影响不大。     

中国莱姆病螺旋体rRNA基因多态性分析

本文作者首次应用地高辛标记的大肠杆菌１６＋２３ＳｒＲＮＡ基因探针分析中国莱姆病螺旋体ｒＲＮＡ基因限制性图谱。４５株中国菌株和６株国外菌株（美国Ｂ３１，２９７；法国２００４７；瑞士ＶＳ４６１；德国ＬＷ２和俄罗斯菌株ＩＰ２ｌ）用于实验，选用ＥｃｏＲⅤ和ＨｉｎｄⅢ两种限制性内切酶。实验结果表明，中国菌株遗传差异性较大，分属三个基因种，即ＧｅｎｏｓｐｅｃｉｅｓⅠ（Ｂ．ｂｕｒｇｄｏｒｆｅｒｉＳｅｎｓｕＳｔｒｉｃｔｏ），ＧｅｎｏｓｐｅｃｉｅｓⅡ（Ｂ．ｇａｒｉｎｉ）和ＧｅｎｏｓｐｅｃｉｅｓⅢ（Ｂ．ａｆｚｅｌｉ），其中Ｂ．ｂｕｒｇｄｏｒｆｅｒｉＳｅｎｓｕＳｔｒｉｃｔｏ基因种在亚洲菌株中是首次发现。此外还发现中国菌株ＨＢ１８的ｒＲＮＡ基因杂交图谱与国内外其它菌株不同。分属于三个基因种的中国菌株的ｒＲＮＡ基因杂交图谱与欧洲和美国菌株略有差异。中国菌株ＨＢ１８可能属于一个新的基因种。     

利用16S rRNA的PCR法鉴定双歧杆菌

随着以双歧杆菌为主的微生态制剂在医疗、保健、食品等行业的广泛应用 ,建立特异、灵敏、快速、简易的双歧杆菌分类鉴定方法日益引起人们的关注。荧光原位杂交〔1〕、ＰＣＲ〔2〕等已被引入双歧杆菌的鉴定。双歧杆菌各种型间发生学上密切相关 ,其 16ＳｒＲＮＡ相似性大于 93 % 〔3〕,这就为直接利用这些序列设计属特异性引物或寡核苷酸探针检测双歧杆菌提供了依据。本研究利用双歧杆菌 16ＳｒＲＮＡ基因序列设计属特异性引物 ,通过ＰＣＲ鉴定双歧杆菌 ,通过琼脂糖凝胶电泳和Ｓｏｕｔｈｅｒｎ杂交法对扩增产物进行特异性及灵敏度方面的研究。…     

用16S rRNA探针定量测定普氏立克次体在细胞内的繁殖量

目的以普氏立克次体为研究对象，建立一种定量测定胞内微生物繁殖的方法。方法从立克次体感染细胞提取总ＲＮＡ为样品或用硫氰酸胍裂解感染细胞，以裂解物为样品，用３２Ｐ标记的１６ＳｒＲＮＡ特异探针做ＲＮＡ酶保护分析。根据探针分子结合数，计算出检测到的靶序列分子数，反映胞内微生物的繁殖量。结果从０．５μｌ直接裂解物可检测到３．９４×１０４个１６ＳｒＲＮＡ分子；从６ｐｇ的总ＲＮＡ中可检测到５．８２×１０４个１６ＳｒＲＮＡ分子。用本法试测立克次体在宿主细胞内的生长曲线，只表现出迟缓期及对数生长期，而无稳定期及衰退期。结论用特异性探针通过ＲＮＡ酶保护分析法可以定量测定胞内微生物在宿主细胞内的繁殖量。由本法测定到的普氏立克次体生长曲线表明难以沿用细菌的生长曲线来描述胞内微生物在宿主细胞内的生长过程     

Identification of an alkaline-tolerant cyclodextrin-metabolizing bacterium and characterization of its cyclodextrinase gene

Bacillus circulans A11, an alkaline-tolerant cyclodextrin-metabolizing bacterium isolated from South-East Asian soil, was reidentified as Paenibacillus sp. A11 based on 16S rRNA gene sequence comparison, G + C content and cellular fatty acid composition. Levels of similarity of the 16S rRNA gene between strain A11 and the Paenibacillus species were 90-99%, while similarity with Bacillus circulans was only 86%. The major cellular fatty acid was anteiso-C15:0 which accounted for 59.3% of the total cellular fatty acids and the G+C content was 50.3 mol%. The CDase gene coding for this enzyme was cloned into E. coli. The open reading frame of the CDase gene was 1,959 bp encoding a CDase of 653 amino acid residues. At maximum growth, the specific activity of the recombinant CDase from E. coli was higher than that of Paenibacillus sp. A11. By SDS-PAGE, the translation product of the recombinant gene showed the same mobility as the purified CDase from the original strain. CDase from both Paenibacillus sp. A11 and E. coli produced glucose and maltose as dominant end-products of beta-CD hydrolysis. The ratio of maltose to glucose was 1:2.     

Analysis of secA1 gene sequences for identification of Nocardia species

Molecular methodologies, especially 16S rRNA gene sequence analysis, have allowed the recognition of many new species of Nocardia and to date have been the most precise methods for identifying isolates reliably to the species level. We describe here a novel method for identifying Nocardia isolates by using sequence analysis of a portion of the secA1 gene. A region of the secA1 gene of 30 type or reference strains of Nocardia species was amplified using secA1-specific primers. Sequence analysis of 468 bp allowed clear differentiation of all species, with a range of interspecies similarity of 85.0% to 98.7%. Corresponding 16S rRNA gene sequences of a 1,285-bp region for the same isolates showed a range of interspecies similarity of 94.4 to 99.8%. In addition to the type and reference strains, a 468-bp fragment of the secA1 gene was sequenced from 40 clinical isolates of 12 Nocardia species previously identified by 16S rRNA gene sequence analysis. The secA1 gene sequences of most isolates showed >99.0% similarity to the secA1 sequences of the type or reference strain to which their identification corresponded, with a range of 95.3 to 100%. Comparison of the deduced 156 amino acid sequences of the SecA1 proteins of the clinical isolates showed between zero and two amino acid residue differences compared to that of the corresponding type or reference strain. Sequencing of the secA1 gene, and using deduced amino acid sequences of the SecA1 protein, may provide a more discriminative and precise method for the identification of Nocardia isolates than 16S rRNA gene sequencing.     

Ehrlichia chaffeensis, a new species associated with human ehrlichiosis.

The bacterial 16S rRNA genes from blood samples of two patients with human ehrlichiosis and from an isolate recovered from one of the patients were amplified by using the polymerase chain reaction. The amplimers were then cloned and sequenced. The 16S rRNA gene sequence was also determined for Ehrlichia canis (two strains), E. equi, E. phagocytophila (two strains), and E. sennetsu (two strains). These sequences, along with a previously published 16S rRNA gene sequence of E. risticii, were compared. The 16S rRNA gene sequences were identical for all three sources of the human ehrlichiosis agent. The sequence comparisons indicate that the human ehrlichiosis agent is a new species most closely related to E. canis (98.2%) and more distantly related to other Ehrlichia spp. We propose that this species be named Ehrlichia chaffeensis sp. nov., with the Arkansas strain as the type strain.     

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.     

Detection of 16S rRNA gene for rapid identification of bacterial pathogens causing peritonitis in patients on continuous ambulatory peritoneal dialysis

PurposePeritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD.MethodsFifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate.ResultsAmong the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed.ConclusionThe molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.     

安徽黄山地区中华硬蜱及其宿主感染莱姆病Borrelia afzelii的检测

利用PCR和病原体分离技术对安徽黄山地区中华硬蜱及其宿主感染莱姆病螺旋体的状况进行了检测,结果从40只黄山林区的中华硬蜱中获得了一株莱姆病螺旋体分离株,寄主动物及其他中华硬蜱均未获得阳性分离结果.以16s rRNA为靶标进行PCR检测,6.78%雌性和5%雄性中华硬蜱发现有阳性感染,2只草兔(92只)和1只麂子(16只)也发现有阳性感染;若蜱和其余宿主动物未发现阳性感染.阳性分离株和扩增获得的168 rRNA序列结果一致,同GenBank中的B.afzelii的系统发育关系最近,提示该分离株螺旋体属于B.afzelii.该结果与前期的实验传播结果提示中华硬蜱是我国南方莱姆病的传播媒介.     

A new agent of mycobacterial lymphadenitis in children: Mycobacterium heidelbergense sp. nov.

Nontuberculous mycobacterial lymphadenitis presents an increasing clinical problem in immunocompetent young children. A slowly growing, nonphotochromogenic mycobacterium was recovered twice (isolates 2553/91 and 2554/91) from the lymphatic tissue of a child with recurrent cervical lymphadenitis. It could be differentiated biochemically from described Mycobacterium species, although it most closely resembled Mycobacterium malmoense by thin-layer chromatography and high-performance liquid chromatography of mycolic acids. A striking characteristic of the isolate was its high degree of susceptibility to antituberculous drugs in vitro, including isoniazid. Direct determination of the 16S rRNA gene sequence revealed a unique sequence and positioned the strain phylogenetically on a branch separate from M. malmoense within a group of slowly growing mycobacteria that show a high degree of similarity to M. simiae at the 16S rRNA gene level. Despite 99.6% sequence identity with M. simiae at the 16S rRNA gene level, DNA-DNA hybridization studies (hydroxyapatite method) demonstrated DNA relatedness of less than 40%. We conclude that this organism is a new species for which we propose the name M. heidelbergense. A culture of the type strain, strain 2554/91, has been deposited in the American Type Culture Collection as strain ATCC 51253.     

Genetic diversity of endophytic bacteria which could be find in the apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants

The genetic diversity of 29 endophytic bacterial strains isolated from apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants grown in Cuba was analysed by Two Primers-Ramdom Amplified Polymorphic DNA fingerprinting (TP-RAPD) and 16S rRNA gene sequencing. The strains were distributed into 17 groups on the basis of their TP-RAPD patterns, and a representative strain from each group was subjected to 16S rRNA gene sequencing. Analysis of these sequences showed that the isolates belong to a wide variety of phylogenetic groups being closely related to species of genera Bacillus and Staphylococcus from Firmicutes, Microbacterium, Micrococcus and Kokuria from Actinobacteria, Rhizobium and Gluconacetobacter from alpha -Proteobacteria, Comamonas and Xanthomonas from beta-Proteobacteria, and Acinetobacter and Pantoea from gamma-Proteobacteria. These results show the complexity of the bacterial populations present in inner tissues of sugarcane, and indicate the interest and relevance of the studies on microbial diversity to improve our knowledge on the plant endophytic bacterial communities.     

Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.     

Parallel Detection of Bacterial Pathogens in Cerebrospinal Fluid with 16S rRNA Probe Microarray

The most commonly used method for detection ofpathogenic bacteria in cerebrospinal fluid (CSF) speci mens of clinical laboratories is isolation and identificationof the causative agents by cultural method, biochemicaland serological t…