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Sample records for 16s rdna gene

  1. Phylogenetic analysis of Thai oyster (Ostreidae) based on partial sequences of the mitochondrial 16S rDNA gene

    DEFF Research Database (Denmark)

    Bussarawit, Somchai; Gravlund, Peter; Glenner, Henrik;

    2006-01-01

    Ten oyster species of the family Ostreidae (Subfamilies Crassostreinae and Lophinae) from Thailand were studied using morphological data and mitochondrial 16S rDNA gene sequences. Additional sequence data from five specimens of Ostreidae and one specimen of Tridacna gigas were downloaded from Gen...

  2. Evolutionary relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA gene sequences.

    Science.gov (United States)

    Dowton, M; Austin, A D; Antolin, M F

    1998-05-01

    Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA gene sequence data. Analyses recovered the few well-supported relationships evident in this family from morphological analyses, viz the monophyly of the microgastroid complex of subfamilies, the monophyly of the cyclostome complex of subfamilies (= braconoids), a sister-group relationship between the Alysiinae and Opiinae, and a close relationship between the Helconinae and Blacinae. With respect to the braconoid complex of subfamilies, a sister-group relationship was recovered between Aphidiinae and Mesostoinae, and a clade composed of Gnamptodontinae + Histeromerinae + Rhyssalinae + Aphidiinae + Mesostoinae was also recovered. The Doryctinae and Rogadinae sensu lato (s.l.) were generally not resolved as monophyletic. With respect to the helconoid complex of subfamilies, a sister-group relationship was recovered between Sigalphinae and Agathidinae, whereas Neoneurinae fell out among other helconoid subfamilies. Other relationships among the helconoid subfamilies were unclear from these analyses. With respect to the microgastroid complex of subfamilies, our data conform to morphological estimates, recovering ((Microgastrinae + Miracinae) + Cardiochilinae) + Cheloninae. The topology of our trees suggests that the cyclostome subfamilies are a natural derived group, inferring that endoparasitism (not ectoparasitism) is the ancestral state for the Braconidae, unless all of the ectoparasitic ancestors of the helconoid + microgastroid subfamilies are now extinct.

  3. Rapid diagnosis of virulent Pasteurella multocida isolated from farm animals with clinical manifestation of pneumonia respiratory infection using 16S rDNA and KMT1 gene

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    Gamal Mohamedin Hassan

    2016-01-01

    Full Text Available Objective: To characterize intra-isolates variation between clinical isolates of Pasteurella multocida (P. multocida isolated from sheep, cattle and buffalo at molecular level to check the distribution of pneumonia and hemorrhagic septicemia in some regions of Fayoum, Egypt. Methods: These isolates were obtained from various locations in the Fayoum Governorate, Egypt and they were identified by amplifying 16S rDNA and KMT1 genes using their DNA as a template in PCR reaction. Results: The results demonstrated that the five selective isolates of P. multocida had similar size of PCR products that generated one band of 16S rDNA having 1 471 bp and KMT1 gene having 460 bp. The phylogenetic tree and similarity of the five selective isolates of P. multocida which were collected from GenBank database were calculated and analyzed for the nucleotide sequence of 16S rDNA and KMT1 genes. The sequencing result of 16S rRNA gene product (1 471 bp for the five selective isolates of P. multocida showed that the isolates of sheep (FUP2 shared 94.08%, 88.10% homology with the buffalo isolate (FUP8 and cattle isolate (FUP9 respectively, whereas, the buffalo isolate (FUP5 shared 98.18% and 94.40% homology with the cattle isolates (FUP12 and FUP9. Conclusions: The results indicated the relationships of P. multocida isolated from buffalo and cattle rather than the close relationships between P. multocida isolated from cattle and sheep. Diagnosis of P. multocida by 16S rDNA and KMT1 gene sequences was important to determine the antigen that is responsible for protective cover within the same group of animals and to help for the production of new vaccines for the control of microbial infection for domestic animals.

  4. Identification of novel spp. of rice and wheat endophytic diazotrophs by 16S rDNA gene and FTIR analysis

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    Mohammad Javad Mehdipour Moghaddam

    2012-06-01

    Full Text Available In this research, six isolates, including three from three rice roots (PxR1, PxR2 and StR1 and three from three wheat roots (PxW1, PxW2 and PxW3 were isolated as endophytic bacteria and except for StR1, all the isolates were identified as Pseudoxanthomonas based on phenotypic analysis including FTIR and PCR amplification of 16S rDNA. The results showed that PxR1, PxR2, PxW1 and PxW2 were all similar and belonged to a novel species of Pseudoxanthomonas, but PxW3 was from different species. StR1 belonged to a novel species of Stenotrophomonas. Two strains including Azospirillum brasiliense Sp7 (S1 and Azospirillum lipoferum (S2 were selected as standard strains and compared with those isolates however, phenotypic and genotypic analysis verified that those isolates were not Azospirillum. For the first time, it was indicated that Pseudoxanthomonas existed as an endophytic bacterium in rice root.

  5. Molecular phylogeny of the butterfly tribe Satyrini (Nymphalidae: Satyrinae) with emphasis on the utility of ribosomal mitochondrial genes 16s rDNA and nuclear 28s rDNA.

    Science.gov (United States)

    Yang, Mingsheng; Zhang, Yalin

    2015-07-09

    The tribe Satyrini is one of the most diverse groups of butterflies, but no robust phylogenetic hypothesis for this group has been achieved. Two rarely used 16s and 28s ribosomal and another seven protein-coding genes were used to reconstruct the phylogeny of the Satyrini, with further aim to evaluate the informativeness of the ribosomal genes. Our maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) analyses consistently recovered three well-supported clades for the eleven sampled subtribes of Satyrini: clade I includes Eritina and Coenonymphina, being sister to the clade II + clade III; clade II contains Parargina, Mycalesina and Lethina, and the other six subtribes constitute clade III. The placements of the taxonomically unstable Davidina Oberthür and geographically restricted Paroeneis Moore in Satyrina are confirmed for the first time based on molecular evidence. The close relationships of Callerebia Butler, Loxerebia Watkins and Argestina Riley are well-supported. We suggest that Rhaphicera Butler belongs to Lethina. The partitioned Bremer support (PBS) values of MP analysis show that the 16s rDNA contributes well to the nodes representing all the taxa from subtribe to species levels, and the 28s rDNA is informative at the subtribe level. Furthermore, our ML analyses show that the ribosomal genes 16s rDNA and 28s rDNA are informative, because most node support values are lower in the ML tree after the removal of them than that in ML tree constructed based on the full nine-gene dataset. This indicates that some other ribosomal genes should be tentatively used through combining with traditionally used protein-coding genes in further analysis on phylogeny of Satyrini, providing that proper representatives are sampled.

  6. Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

    Science.gov (United States)

    Hayashi, K; Tazumi, A; Nakanishi, S; Nakajima, T; Matsubara, K; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

    2012-06-01

    Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

  7. Phylogenetic relationships among the microgastroid wasps (Hymenoptera: braconidae): combined analysis of 16S and 28S rDNA genes and morphological data.

    Science.gov (United States)

    Dowton, M; Austin, A D

    1998-12-01

    Relationships among the microgastroid complex of braconid wasps were investigated using sequence data from the 16S mitochondrial rDNA and 28S (D2 expansion region) nuclear rDNA genes, as well as morphological data. Parsimony analysis of these gene fragments, both separately and combined, indicated that Neoneurus (Neoneurinae) and Ichneutes (Ichneutinae) were no more closely related to the microgastroids than were a range of helconoid taxa. Combined parsimony analysis of the microgastroids indicated the relationships ((Cardiochilinae + Microgastrinae) + Miracinae) + Cheloninae, with Adeliinae falling inside the Cheloninae. Bootstrap proportions for each of these nodes were greater than 70%. Character reweighting (sensu Farris), using the rescaled consistency index, also recovered these relationships. Mapping of lifestyle traits onto this relatively well supported phylogeny indicated that solitary endoparasitism is ancestral for the microgastroids, with a single origin for egg-larval endoparasitism in the Cheloninae + Adeliinae. Mapping of the radiation of the microgastroids into lepidopteran hosts was less clear, due to the specialized biology of the most basal microgastroid clade, the Cheloninae + Adeliinae. Our data are consistent with attack of concealed lepidopteran hosts as the plesiomorphic lifestyle, at least for the Miracinae + Cardiochilinae + Microgastrinae, with radiation into more exposed hosts in the Cardiochilinae + Microgastrinae.

  8. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

    Science.gov (United States)

    Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

    2008-10-01

    In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

  9. Toxicity analysis of pesticides on cyanobacterial species by 16S rDNA molecular characterization

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    J. I. Nirmal Kumar

    2013-06-01

    Full Text Available Damaging effects of endosulfan on native structure of DNA, evident as a result of PCR based assay such as 16S rDNA amplification and sequencing, led to formation of gaps, mismatching of base pairs and dissimilarities in entire 16S rDNA sequences of treated cultures. Endosulfan was the most fatal to Westiellopsis prolifica of 16S rDNA region at 40ppm insecticide induced series of mispairing, and other lesions amounting up to 20% dissimilarity and 7% gaps. Whereas, 16S rDNA region of Anabaena fertilissima was comparatively less influenced with 18% dissimilarity and 7% gaps in response to 12ppm endosulfan, while 16S rDNA gene of Aulosira fertilissima was the least prone to changes with 17% dissimilarity, and 5% gaps under 60ppm endosulfan stress by the end of 16 days. On the other side, impact of fungicide tebuconazole after 16 days reflected identities up to 78% and 8% gaps for 30ppm treated A. fertilissima, while 60ppm treatment instilled 79% similarities with 10% gaps in W. prolifica and 83% identities with 5% gaps of Aulosira fertilissima after 16 days.

  10. Phylogenetic and characteristic analysis of 16S rDNA and rpoB gene sequence of Klebsiella%克雷伯菌16S rDNA和rpoB基因系统进化及序列特征分析

    Institute of Scientific and Technical Information of China (English)

    郭晓琳; 王多春; 阚飙; 张燕敏; 张怡; 左颖; 魏来; 高燕

    2009-01-01

    identified as genus Klebsiella (with 15 of K. Pneumoniae and 3 of K. Oxytoca) by automated biochemical tests were selected. DNA were extracted, 16S rDNA and rpoB genes were amplified and sequenced with Klebsiella 16S rDNA and rpoB primers. Together with already published 8 species of Klebsiella and 9 species of Enterobacteriaceae 16S rDNA and rpoB sequences from GenBank, totally 35 sequences of 16S rDNA and rpoB respectively, phylogenetic trees were constructed with MEGA 4.0 to the analysis of groups. DNAStar/MegAlign was used for comparison of variable regions of 16S rDNA and rpoB, with analysis of degree of divergent at the same time. Results As for all 35 sequences, both 16S rDNA and rpoB phylogenetic trees divided Klebsiella species into three groups, 15 of K. Pneumoniae in this study and 6 of K. Pneumoniae from GenBank (except for K. Oxytoca and K. Mobilis) cluster to group Ⅰ, K. Oxytoca and K. Mobilis were cluster to group Ⅱ and Ⅲ, respectively. In rpoB phylogenetic tree, no matter group Ⅰ and group Ⅱ, or subgroup within group Ⅰ, the bootstrap values in each node of rpoB phylogenetic tree is obviously higher than that of 16S rDNA. Moreover, as for cluster to K. Oxytoca, rpoB is better than 16S rDNA. Analysis nucleic acid sequences of Klebsiella species, with 41 variable regions and 4 most significant regions were found within the Klebsiella 16S rDNA, while rpoB with 63 variable regions, and 1 most significant region. The similarity of 16S rDNA and rpoB within Klebsiella were 95.9%-100% and 90.2%-100% respectively. Further analysis divergent degree of 16S rDNA and rpoB within Klebsiella, the divergent value of rpoB (0-10.6) is higher than that of the 16S rDNA(0-4.0). Conclusion As for molecular classification and identification within KlebsieUa species, rpoB has more advantages than 16S rDNA.

  11. Identificarion of contaminant bacteria in cachaça yeast by 16s rDNA gene sequencing Identificação de bactérias contaminantes de fermento de cachaça por seqüenciamento do gene 16s rDNA

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    Osmar Vaz de Carvalho-Netto

    2008-01-01

    Full Text Available Cachaça is a typical Brazilian liquor produced from the distillation of fermented sugarcane juice mainly by Saccharomyces cerevisiae. Most of the domestic production is artisanal, and producers usually are not concerned regarding microbiological control of the fermentation. This study aimed to characterize the contaminant bacterial community of the yeast used in the production of cachaça in an artisanal still. Four samples were collected, of which one (NA was used for comparison purposes and was collected one year earlier. The remaining samples were collected at three different periods: at the end of the first day of fermentation (NP, after fifteen days (NS, and thirty days after the same yeast was used (NT. Five hundred and eighty-seven sequences were analyzed from the partial sequencing of the 16S rDNA gene. Sequence analyses revealed the presence of 170 operational taxonomic units (OTUs. Of these, only one was shared among three samples and seventeen were shared between two samples. The remaining 152 OTUs were identified only once in distinct samples indicating that the contaminant bacterial population is highly dynamic along the fermentation process. Statistical analyses revealed differences in bacterial composition among samples. Undescribed species in the literature on yeasts of cachaça were found, such as Weissella cibaria, Leuconostoc citreum, and some species of Lactobacillus, in addition to some unknown bacteria. The community of bacteria in the fermentation process is much more complex than it was previously considered. No previous report is known regarding the use of this technique to determine bacterial contaminants in yeast for the production of cachaça.A cachaça é uma bebida típica brasileira produzida a partir da destilação do caldo de cana-de-açúcar fermentado principalmente por Saccharomyces cerevisiae. Grande parte da produção nacional é artesanal, e não há uma preocupação por parte dos produtores quanto ao

  12. Identification of Lactic Acid Bacteria Ⅱ 32 by Sequence Analysis of 16S rDNA and RecA - gene%16S rDNA和recA-gene对乳酸菌Ⅱ32的鉴定

    Institute of Scientific and Technical Information of China (English)

    刘长建; 权春善; 范圣第

    2007-01-01

    对乳酸菌Ⅱ32进行了生化实验.以菌株Ⅱ32的总DNA为模板,采用细菌通用的引物,对其16S rDNA进行特异扩增,并进行序列测定,将测定结果与GenBank DNA数据库中已知菌种的16S rDNA序列通过BLAST软件进行分析比较,初步确定该菌株为戊糖乳酸菌、植物乳杆菌或类植物乳杆菌.采用recA-gene约300bp的特异扩增片段最终确定乳酸菌Ⅱ32为类植物乳杆菌.

  13. Identidade molecular dos fitoplasmas associados aos enfezamentos do tomateiro e da berinjela com base na análise do gene 16S rDNA Molecular identity of the phytoplasma associated to stunting of tomato and eggplant on the basis of analyses of the 16S rDNA

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    Ana Paula de Oliveira Amaral Mello

    2007-09-01

    , shortening internodes, reduced size of leaves, flowers and fruits were observed. In nested PCR with primers R16 mF1/mR2 e R16 F2n/R2, fragments of 1.2kb in size were amplified from symptomatic samples demonstrating the presence of phytoplasmas in plant tissues. By using especific primers pairs it was demonstrated that the phytoplasmas were affiliated to group 16SrIII. RFLP analysis using the restriction enzymes AluI, HpaII, KpnI, MboI, MseI, and RsaI confirmed that the phytoplasmas were representatives of group 16SrIII. Amplified DNA fragments were cloned in Escherichia coli, sequenced and compared by sequence similarities among themselves and with sequences belonging to phytoplasmas of group 16SrIII. Sequence similarities greater than 95% were found when the phytoplamas detected in tomato and eggplant were compared to the representatives of group 16SrIII. Values of 98-99% were obtained when sequences of phytoplasmas found in tomato and eggplant were compared among themselves. The results evidenced that tomato and eggplant stunting were associated with the same phytoplasma based upon the sequencing of the 16S rDNA gene.

  14. Detection of 16S rDNA and PIA genes of Neisseria isolated from the patients with male genitourinary tract infections%男性泌尿生殖道感染患者分离奈瑟菌的16S rDNA和PIA基因检测

    Institute of Scientific and Technical Information of China (English)

    姜敏敏; 王涛; 王和

    2012-01-01

    目的 检测男性泌尿生殖道分离奈瑟菌的16S rDNA和PIA基因,探讨奈瑟菌属菌种的基因鉴定及其致病机理.方法 用PCR扩增和核苷酸序列分析方法分别检测11例男性泌尿生殖道感染患者泌尿生殖道分离的14株奈瑟菌属菌种的16S rDNA和PIA基因及其序列.结果 14株奈瑟菌属菌种经16S rDNA检测鉴定分别为淋病奈瑟菌2株,黏液奈瑟菌3株,灰色奈瑟菌5株,微黄奈瑟菌2株,干燥奈瑟菌1株,嗜乳糖奈瑟菌及多糖奈瑟菌各1株;与常规细菌学方法鉴定的符合率为85.7%.非淋球菌奈瑟菌未检出淋病奈瑟菌毒力相关的PIA核苷酸序列.结论 常规细菌学方法与染色体16S rDNA检测及其序列分析方法的联合使用,可提高奈瑟菌属菌种感染的实验室诊断准确率;PIA基因对于奈瑟菌属的男性生殖道致病性无关.%Objective To detect the 16S rDNA and PIA genes of Neisseria isolated from the patients with male genitourinary tract infections and investigate the gene identification and pathogenesis of Neisseria species. Methods The 16S rRNA and PIA genes and their sequences of 14 Neisseria species isolated from reproductive tract of 11 patients with male genitourinary tract infections were identified by PCR amplification and nucleotide sequencing. Results Fourteen Neisseria strains contained 2 strains of Neisseria gonorrhoeae, 3 strains of Neisseria mucosa, 5 strains of Neisseria cinerea, 2 strain of Neisseria subflava, 1 strain of Neisseria sicca,1 strain of Neisseria lactamica and 1 strain of Neisseria polysaccharea. The accordance rate of the results reported by 16S rDNA and routine bacteriological method was 85. 7%. The nucleotide sequence of virulence-associated gene PIA of Neisseria gonorrhoeae was not found in non-gonococcal strains of Neisseria. Conclusion The accuracy rate of laboratory diagnosis for the infection caused by Neisseria can be improved by combined use of routine bacteriological method and 16S rDNA

  15. Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

    Science.gov (United States)

    Trcek, Janja

    2005-10-01

    Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for

  16. Genetic analysis on 16S rDNA of brucella%布鲁菌16S rDNA基因遗传分析

    Institute of Scientific and Technical Information of China (English)

    李克诚; 周邦谣; 夏菲

    2013-01-01

    目的 通过分析临床分离的布鲁菌、其他盐杆菌科细菌以及临床常见致病菌的16S rDNA基因片段的差异并构建16S rDNA系统发育树,为进一步研究布鲁菌打下基础.方法 PCR扩增临床分离株的16SrDNA并测序;从EMBL下载常见盐杆菌科细菌和临床上常见致病菌的16S rDNA序列.利用CLUSTALX和MEGA程序进行16S rDNA比对并构建系统发育树.结果 成功扩增了临床分离菌株的16S rDNA,得到测序结果,比对临床分离菌株和相关菌株后,发现了特异序列5’-ATCCCGGTCGCGGTTAGTGG-3';系统发育树表明不同种的布鲁菌间的距离非常接近;布鲁菌和醋菌属的进化距离较近,但是和其他的盐杆菌的进化距离较远.结论 在布鲁菌16S rDNA中存在特异序列5'-ATCCCGGTCGCGGTTAGTGG-3’,有可能作为探针来快速检测布鲁氏菌.%Objective To analyze and to compare the genetic characteristics of 16S rDNA gene of Brucella with other halobacteriaceae and pathogenic bacteria, and to construct phylogenetic tree to find the specific sequence. Methods 16s rDNA of Brucella isolated from clinical sample was amplified and sequenced, which was then compared with sequences of halobacteriaceae and other pathogenic bacteria downloaded from EM-BL. CLUSTALA and MEGA software were used for the comparison of the sequences and building of the phylogenetic tree. Results 16s rDNA was successfully amplified and sequenced. Meanwhile, a specific sequence of 5' -ATCCCGGTCGCGGTTAGTGG-3' was identified. Phylogenetic tree showed that the distance was very close among different species of Brucella and was also closer to acetobacter sp. . but was farther to other halobacteriaceae. Conclusions A specific sequence is present in 16S rDNA of brucella which could be used as a probe to detect Brucella.

  17. 稻纵卷叶螟感染Wolbachia的ftsZ基因和16S rDNA基因的序列分析%Sequence analysis of ftsZ and 16S rDNA genes of Wolbachia in Cnaphalocrocis medinalis (Guenée) ( Lepidoptera: Pyralidae)

    Institute of Scientific and Technical Information of China (English)

    柴换娜; 杜予州; 吴海燕

    2011-01-01

    Wolbachia is a group of intracellular inherited endosymbiontic bacteria infecting a wide range of arthropods and other animals. The infection status of Wolbachia in the migratory pest Cnaphalocrocis medinalis (Guenée) was studied to provide the basis for revealing the reproductive manipulation mechanism and transmission mechanism of Wolbachia in this pest. In this study, specific primers derived fromftsZ and 16S rDNA genes were used to amplify DNA of Wolbachia from 20 populations of C. medinalis in China by polymerase chain reaction (PCR). The results indicated that the C. medinalis populations in China were widely infected by Wolbachia, the highest infection rate was 90% in Wenzhou of Zhejiang and Yangzhou of Jiangsu, and the lowest rate was 40% in Ya' an of Sichuan, Changsha of Hunan and Ninghe of Tianjin.The ftsZ sequences and 16S rDNA sequences were exactly the same in all positive samples from different regions. Wolbachia ftsZ sequences and 16S rDNA sequences in C. medinalis showed 99% - 100% and 98% -99% similarity with others belonging to Group B, respectively, suggesting that Wolbachia in C.medinalis belong to Group B. The results show that the infection type of Wolbachia in the C. medinalis is relatively ingle. This is the first report that Wolbachia is distributed in the populations of C. maedinalis in China.%Wolbachia是一类胞质遗传的内共生菌,广泛分布于节肢动物和其他动物中,与宿主的生殖调控密切相关.通过研究迁飞性害虫稻纵卷叶螟Cnaphalocrocis medinalis(Guen e)的Wolbachia感染情况,为探讨Wolbachia在迁飞性昆虫中的生殖调控和传递方式等提供基础资料.本研究应用Wolbachia的ftsZ基因和16S rDNA基因的特异性引物,通过PCR扩增的方法对我国20个地区的稻纵卷叶螟样本进行了检测.结果表明:中国不同地区的稻纵卷叶螟感染Wolbachia的现象较为普遍,其中浙江温州和江苏扬州样本的感染率最高(90%);四川雅安、湖

  18. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae).

    Science.gov (United States)

    Nava, Santiago; Venzal, José M; Labruna, Marcelo B; Mastropaolo, Mariano; González, Enrique M; Mangold, Atilio J; Guglielmone, Alberto A

    2010-08-01

    We supply information about hosts and distribution of Amblyomma dubitatum. In addition, we carry out an analysis of genetic divergence among specimens of A. dubitatum from different localities and with respect to other Neotropical Amblyomma species, using sequences of 16S rDNA gene. Although specimens of A. dubitatum were collected on several mammal species as cattle horse, Tapirus terrestris, Mazama gouazoubira, Tayassu pecari, Sus scrofa, Cerdocyon thous, Myocastor coypus, Allouata caraya, Glossophaga soricina and man, most records of immature and adult stages of A. dubitatum were made on Hydrochoerus hydrochaeris, making this rodent the principal host for all parasitic stages of this ticks. Cricetidae rodents (Lundomys molitor, Scapteromys tumidus), opossums (Didelphis albiventris) and vizcacha (Lagostomus maximus) also were recorded as hosts for immature stages. All findings of A. dubitatum correspond to localities of Argentina, Brazil, Paraguay and Uruguay, and they were concentrated in the Biogeographical provinces of Pampa, Chaco, Cerrado, Brazilian Atlantic Forest, Parana Forest and Araucaria angustifolia Forest. The distribution of A. dubitatum is narrower than that of its principal host, therefore environmental variables rather than hosts determine the distributional ranges of this tick. The intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil and Uruguay was in all cases lower than 0.8%, whereas the differences with the remaining Amblyomma species included in the analysis were always bigger than 6.8%. Thus, the taxonomic status of A. dubitatum along its distribution appears to be certain at the specific level.

  19. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    Science.gov (United States)

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.

  20. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    Science.gov (United States)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  1. Comparing the Potential for Identification of Lactobacillus spp. of 16S rDNA Variable Regions

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    Diego Mauricio Riaño-Pachón

    2013-07-01

    Full Text Available 0 0 1 248 1368 Universidad de los Andes 11 3 1613 14.0 Normal 0 21 false false false ES-TRAD JA X-NONE 16s rDNA is used for bacterial identification because its variation rate between species allows differentiation. The gene for this ribosomal subunit has 9 variable regions and some of them give more information than others. We were interested in evaluating the potential for species identification of each region and their combinations. We extracted the V1 to V8 regions of 16s rDNA from different strains and species of Lactobacillus and analyzed them using STAP (ss-RNA Taxonomy Assigning Pipeline and RDP (Ribosomal Database Project multiclassifier packages. Phylogenetic trees obtained by maximum likelihood analyses were compared. Classification results show that many regions give the correct genus classification using RDP and STAP, however they are not enough to classify up to the level of species. V5V6 region presents the highest quantity of informative fragments but also present the highest rate of false negatives. V1V3 region presents the highest rate of true positives (species using STAP and the region V5V8 in RDP (genus.The phylogenetic result shows that the reference topology could be obtained using different combination of regions as V1V3 and V1V8.The experimental validation was done using commercial strains from a probiotic tampon. Sequencing analysis show that the V1V3 region gives the same information and result as the complete 16s rDNA; the three isolated strains correspond to the strains indicated in the product. We conclude that the V1V3 region is the minimum required region to classify Lactobacillus spp. in the correct way and this region is useful in metagenomics to analyze probiotics samples  COMPARACIÓN ENTRE EL POTENCIAL DE LAS REGIONES VARIABLES DEL 16S rDNA PARA LA IDENTIFICACIÓN DE Lactobacillus spp (LactobacilliaceaeEl 16s rDNA es utilizado para la identificación bacteriana dada su tasa de variaci

  2. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    National Research Council Canada - National Science Library

    Lau, S K P; Ng, K H L; Woo, P C Y; Yip, K-T; Fung, A M Y; Woo, G K S; Chan, K-M; Que, T-L; Yuen, K-Y

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system...

  3. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    Science.gov (United States)

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  4. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    OpenAIRE

    Chan, Km; Yuen, KY; Que, TI; Woo, GKS; Fung, Amy; Yip, KT; Woo, PCY; Ng, KHL; Lau, SKP

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available ...

  5. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    OpenAIRE

    Lau, S.K.P.; Ng, K H L; Woo, P C Y; Yip, K‐t; Fung, A M Y; Woo, G K S; Chan, K‐m; Que, T‐L

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available ...

  6. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    Directory of Open Access Journals (Sweden)

    Ekwan Nofa Wiratno

    2016-04-01

    Full Text Available High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g.L-1, followed by Bacillus methylotrophicus RP 3.2 and B. methylotrophicus RP 7.2 isolate (10.11 g.L-1 and 9.63 g.L-1 respectively. Paenibacillus polymyxa RP 2.2 showed similarity in nucleotide composition (ATGC with B. methylotrophicus RP 3.2, B. methylotrophicus RP 7.2, P. polymyxa CR1, Bacillus amyloliquefaciens NELB-12, and Paenibacillus polymyxa WR-2. Clostridium acetobutylicum ATCC 824 showed similarity in nucleotide composition (ATGC with Clostridium saccharoperbutylacetonicum N1-4, and Clostridium saccharobutylicum Ox29. The lowest G+C content was C. saccharobutylicum Ox29 (51.35%, and the highest was B. methylotrophicus RP 7.2 (55.33%. Conserved region of 16S rDNA (1044 bp were consisted of 17 conserved sequences. The number of Parsimony Informative Site (PIS was 319 spot and single tone was 48 spot. We found in this study that all of butanolproducing bacterial DNA sequences have clustered to 8 haplotypes. Based on the origin of sample, there were three haplotype groups. Bacteria from group A were could produce butanol 8.9-10.34 g.L-1, group B 9.2-14.2 g.L-1 and group C was could produce butanol 0.47 g.L-1. The haplotype analysis of bacteria based on 16S rDNA sequences in this study could predict capability of butanol production.

  7. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    Science.gov (United States)

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  8. Phylogenetic relationships of five species of Dorippinae (Crustacea, Decapoda) revealed by 16S rDNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    FANYu; LIXinzheng; SONGLinsheng; CAIZhonghua

    2004-01-01

    A molecular phylogeny is presented for the subfamily Dorippinae (including 9 individuals, representing 5 species and 4 genera), based on the sequence data from 16S rRNA gene. Two-cluster test between lineages in these phylogenetic trees has been performed. On the basis of rate constancy, the rate of nucleotide substitutions of 16S rDNA sequence data is estimated as 0.27% per million years. The analysis strongly supports the recognition of the Dorippinae as a monophyletic subfamily. Phylogenetic tree indicates that the subfamily Dorippinae is divided into two main clades, and genus Dorippe appears basal in the subfamily, diverging from other species 36.6 Ma ago. It is also clear that the Heikea is closely related to the genus Neodorippe. The divergence time between them is 15.8 Ma.

  9. [PCR rDNA 16S used for the etiological diagnosis of blood culture negative endocarditis].

    Science.gov (United States)

    Baty, G; Lanotte, P; Hocqueloux, L; Prazuck, T; Bret, L; Romano, M; Mereghetti, L

    2010-06-01

    We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

  10. Diversity of 16S rDNA and environmental factor influencing microorganisms in Malan ice core

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The research on extrempholic microorganisms in glacial low-temperature environment receives more attention than ever before. Due to the successive chronological records in ice core, it is important to initiate microbiological studies on ice core samples. 23 samples from one ice core, drilled from central Qinghai-Tibetan Plateau, were analyzed. The number of total microorganisms and culturable microorganisms in different layers showed that it related with the content of dust in ice. It is suggested that the distribution of microorganisms in ice depends on the transportation of materials during glacier development. The bacteria diversity in Malan Glacier was analyzed by 16S rDNA sequencing methods, which showed that many sequences were similar to known psychrophilic bacteria.

  11. Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

    Science.gov (United States)

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.

  12. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

    Directory of Open Access Journals (Sweden)

    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  13. Sequencing of an atypical Brucella strain 16S rDNA%1株非典型布鲁杆菌的16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    刘志国; 王妙; 刘日宏; 崔步云

    2015-01-01

    目的 分析非典型布鲁杆菌的16S rDNA序列,评价16S rDNA序列测定方法鉴定布鲁杆菌的可行性.方法 用常规方法对l株非典型菌株进行初步鉴定;提取菌株的DNA,双向测定16S rDNA全序列,在NCBI对该序列进行Blast比对,用DNAMAN软件进行菌株的16S rDNA序列一致性比对;同时,从GenBank下载与布鲁杆菌有血清学交叉反应菌株的16S rDNA全序列,用MEGA 6.0构建16S rDNA系统发生树.结果 常规鉴定显示,试验菌株为非典型布鲁杆菌.试验菌株的16S rDNA序列与布鲁杆菌基因的相似性为99%,与牛种布鲁杆菌544A、羊种布鲁杆菌16M的16S rDNA序列的一致性为96.99%.系统发生树显示,试验菌株为布鲁杆菌,且与牛种布鲁杆菌544A、羊种布鲁杆菌16M有较为密切的亲缘关系.结论 试验菌株为非典型布鲁杆菌,16S rDNA序列测定是一种简便、快速、准确的非典型布鲁杆菌鉴定方法.%Objective To sequence an atypical Brucella strain 16S rDNA,and to evaluate the feasibility of 16S rDNA sequencing method for identification of Brucella.Methods Preliminary identification of atypical strains was carried out with conventional method.Strain DNA was extracted,and 16S rDNA complete sequence was bidirectional sequenced,and Blast in NCBI and DNAMAN software were used for comparison of the sequence identities of the 16S rDNA.Moreover,16S rDNA complete sequence of the stains those were known to cross-react serologically with Brucella was downloaded from GenBank,MEGA 6.0 was used to construct the phylogenetic tree.Results The conventional identification results revealed that it was an atypical Brucella,the gene similarity between the sequences of the test strain 16S rDNA and Brucella was 99%,between 16S rDNA sequence of Brucella abortus 544A and Brucella melitensis 16M was 96.99%.Phylogenetic tree revealed that the test stain was a Brucella,and closely related to Brucella abortus 544A and Brucella melitensis 16M.Conclusion The

  14. Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides AT+CG in the mitogenome of Kamimuria wangi.

    Science.gov (United States)

    Seligmann, Hervé

    2016-07-01

    Swinger DNAs are sequences whose homology with known sequences is detected only by assuming systematic exchanges between nucleotides. Nine symmetric (XY, i.e. AC) and fourteen asymmetric (X->Y->Z, i.e. A->C->G) exchanges exist. All swinger DNA previously detected in GenBank follow the AT+CG exchange, while mitochondrial swinger RNAs distribute among different swinger types. Here different alignment criteria detect 87 additional swinger mitochondrial DNAs (86 from insects), including the first swinger gene embedded within a complete genome, corresponding to the mitochondrial 16S rDNA of the stonefly Kamimuria wangi. Other Kamimuria mt genome regions are "regular", stressing unanswered questions on (a) swinger polymerization regulation; (b) swinger 16S rDNA functions; and (c) specificity to rDNA, in particular 16S rDNA. Sharp switches between regular and swinger replication, together with previous observations on swinger transcription, suggest that swinger replication might be due to a switch in polymerization mode of regular polymerases and the possibility of swinger-encoded information, predicted in primordial genes such as rDNA.

  15. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    Directory of Open Access Journals (Sweden)

    Julie M. Allen

    2016-07-01

    Full Text Available Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura. We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain.

  16. Detection of transient bacteraemia following dental extractions by 16S rDNA pyrosequencing: a pilot study.

    Directory of Open Access Journals (Sweden)

    Alfonso Benítez-Páez

    Full Text Available OBJECTIVE: The current manuscript aims to determine the prevalence, duration and bacterial diversity of bacteraemia following dental extractions using conventional culture-dependent methods and 16S rDNA pyrosequencing. METHODS: The study group included 8 patients undergoing dental extractions under general anaesthesia. Peripheral venous blood samples were collected at baseline, 30 seconds and 15 minutes after the dental extractions. Blood samples were analysed for bacteraemia applying conventional microbiological cultures under aerobic and anaerobic conditions as well as pyrosequencing using universal bacterial primers that target the 16S ribosomal DNA gene. RESULTS: Transient bacteremia was detected by culture-based methods in one sample at baseline time, in eight samples at 30 seconds, and in six samples at 15 minutes after surgical procedure; whereas bacteraemia was detected only in five blood samples at 30 seconds after dental extraction by using pyrosequencing. By applying conventional microbiological methods, a single microbial species was detected in six patients, and Streptococcus viridans was the most frequently cultured identified bacterium. By using pyrosequencing approaches however, the estimated blood microbial diversity after dental extractions was 13.4±1.7 bacterial families and 22.8±1.1 genera per sample. CONCLUSION: The application of 16S rDNA pyrosequencing underestimated the prevalence and duration of bacteraemia following dental extractions, presumably due to not reaching the minimum DNA required for PCR amplification. However, this molecular technique, unlike conventional culture-dependent methods, revealed an extraordinarily high bacterial diversity of post-extraction bacteraemia. We propose that microorganisms recovered by culture may be only the tip of an iceberg of a really diverse microbiota whose viability and potential pathogenicity should be further studied.

  17. Distribution, hosts, 16S rDNA sequences and phylogenetic position of the Neotropical tick Amblyomma parvum (Acari: Ixodidae).

    Science.gov (United States)

    Nava, S; Szabó, M P J; Mangold, A J; Guglielmone, A A

    2008-07-01

    The hosts, distribution, intraspecific genetic variation and phylogenetic position of Amblyomma parvum (Acari: Ixodidae) have recently been re-assessed. Data on this tick's hosts and distribution were obtained not only from existing literature but also from unpublished records. Sequences of the ticks' mitochondrial 16S ribosomal DNA (rDNA) were used to evaluate genetic variation among specimens of A. parvum from different localities in Argentina and Brazil, and to explore the phylogenetic relationships between this tick and other Amblyomma species. Although several species of domestic and wild mammal act as hosts for adult A. parvum, most collected adults of this species have come from cattle and goats. Caviid rodents of the subfamily Caviinae appear to be the hosts for the immature stages. So far, A. parvum has been detected in 12 Neotropical biogeographical provinces (Chaco, Cerrado, Eastern Central America, Venezuelan Coast, Pantanal, Parana Forest, Caatinga, Chiapas, Venezuelan Llanos, Monte, Western Panamanian Isthmus, and Roraima) but the Chaco province has provided significantly more specimens than any other (P<0.0001). The 16S rDNA sequences showed just 0.0%-1.1% divergence among the Argentinean A. parvum investigated and no more than 0.2% divergence among the Brazilian specimens. The observed divergence between the Argentinean and Brazilian specimens was, however, greater (3.0%-3.7%). Although there is now molecular and morphological evidence to indicate that A. parvum, A. pseudoparvum, A. auricularium and A. pseudoconcolor are members of a natural group, previous subgeneric classifications do not reflect this grouping. The subgeneric status of these tick species therefore needs to be re-evaluated. The 16S-rDNA-based evaluation of divergence indicates that the gene flow between Argentinean and Brazilian 'A. parvum' is very limited and that the Argentinean 'A. parvum' may be a different species to the Brazilian.

  18. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    Science.gov (United States)

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

  19. When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

    Science.gov (United States)

    Budaeva, Nataliya; Schepetov, Dmitry; Zanol, Joana; Neretina, Tatiana; Willassen, Endre

    2016-01-01

    Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected.

  20. Sequencing of 16S rDNA of Klebsiella: taxonomic relations within the genus and to other Enterobacteriaceae.

    Science.gov (United States)

    Boye, Kit; Hansen, Dennis S

    2003-02-01

    The 16S rDNAs of 20 strains of Klebsiella were sequenced and used for construction of a phylogenetic tree together with already published Enterobacteriaceae 16S rDNA sequences. The taxonomy within the Klebsiella genus, as reflected by the 16S rDNA tree, was in agreement with existing DNA-DNA hybridisation and numerical taxonomy data, indicating that for Klebsiella, 16S rDNA sequencing is a valid method for identification and taxonomical purposes. Five closely related clusters were found in the Klebsiella genus; Cluster I, K. oxytoca; Cluster II, K. terrigena, Cluster III, K. planticola and K. ornithinolytica; Cluster IV, Enterobacter aerogenes (K. mobilis); and Cluster V, K. pneumoniae. The position of Calymmatobacterium granulomatis within the genus and closest to K. pneumoniae was confirmed. For the species K. oxytoca, data seem to indicate a subdivision into two subspecies. In addition, a biochemically aberrant Klebsiella strain (BEC441) that was included in the analysis could not be assigned to any of the known species, but was found to be closest related to K. oxytoca. Furthermore, the high sequence similarity between the two environmental species K. planticola and K. ornithinolytica does not justify a distinction of the two species. Finally, within a 165-bp stretch of the 16S rDNA sequences, species-specific nucleotides were found.

  1. Comparison of two approaches for the classification of 16S rRNA gene sequences.

    Science.gov (United States)

    Chatellier, Sonia; Mugnier, Nathalie; Allard, Françoise; Bonnaud, Bertrand; Collin, Valérie; van Belkum, Alex; Veyrieras, Jean-Baptiste; Emler, Stefan

    2014-10-01

    The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

  2. Differentiation of Acidithiobacillus ferrooxidans and A. thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis.

    Science.gov (United States)

    Bergamo, Rogério F; Novo, Maria Teresa M; Veríssimo, Ricardo V; Paulino, Luciana C; Stoppe, Nancy C; Sato, Maria Inês Z; Manfio, Gilson P; Prado, Paulo Inácio; Garcia, Oswaldo; Ottoboni, Laura M M

    2004-09-01

    Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.

  3. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    Science.gov (United States)

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

  4. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

    Science.gov (United States)

    Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

  5. Length polymorphisms for intergenic spacer regions of 16S-23S rDNA in members of the new hydrogen-producing bacteria

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A method based on PCR amplification of the 16S rRNA gene (rDNA) -23S rDNA intergenic spacer regions (ISR) was developed for the identification of species within the novel group hydrogen-producing anaerobes. The sizes of the PCR products varied from 1264 to 398 bp. Strain of isolate Rennanqilyf 3 was characterized as having products of 1262, 398, 638, 437 and 436 bp. The isolate Rennanqilyf 1 had product of 1264 bp. The isolate Rennanqilyf 13 had products of 1261, 579 and 485 bp. Of the 3 species of the novel group hydrogenproducing anaerobes examined, no one was indistinguishable. Two environmental isolates were identified as hydrogen-producing bacteria, which were new species in present taxon. Rennanqilyf 3 could not be associated With any Clostridium sp. Studied. Rennanqilyf 1 could be classified into Clostridium genus. The combination between 16S rDNA equencing and length polymorphisms of IRS in 16S-23S rDNA is a better method for determining species of the hydrogen-producing bacteria.

  6. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  7. 沼泽红假单胞菌16S rDNA PCR快速检测方法研究%Study on the Detection Method of Rhodopseudomonas Palustris with 16S rDNA PCR

    Institute of Scientific and Technical Information of China (English)

    王妍力; 耿亚亚; 郝葆青

    2016-01-01

    Objective:according to the species attributes and highly conservation of 16S rDNA sequence of photosynthetic bacteria, the specific primers were designed, and the PCR reaction con-ditions were optimized, in order to explore the identifying methods to Rhodopseudomonas palustris. Method: the chromosomal DNA was extracted from Rhodopseudomonas palustris cell, and it was iso-lated and purified. The 16S rDNA gene sequences of the Pseudomonas genus, Escherichia coil and Nocardiopsis sp came from the GenBank respectively, and the gene sequence fragments of variable region were analyzed and compared with each other for the design of the species-specific primers. Several factors that may affect the PCR amplification were analyzed, and also some pre-tests were done in order to determine the best conditions for the PCR amplification. And then the PCR ampli-fication was done, the PCR products were cloned and sequenced. Meanwhile morphological and bio-chemical characteristics of Rhodopseudomonas palustris were tested. Conclusion:the method was de-veloped to detect Rhodopseudomonas palustris by PCR amplification with species-specific primers, which had the properties of strong specificity, high sensitivity, good repeatability, high maneuver-ability and low price. The whole process from extracting the DNA to the PCR amplification was about 3 h for identifying the strains of Rhodopseudomonas palustris.%目的:依据反映光合菌物种属性和高度保守性的16S rDNA序列,设计其特异性引物,优化并确定PCR反应条件,探索沼泽红假单胞菌快速鉴别的方法。方法:提取沼泽红假单胞菌菌体染色体DNA,分离和纯化;从GenBank分别获取假单胞菌属、大肠杆菌和诺卡氏菌属的16S rDNA基因序列,分析比较并确定其可变区的基因序列片段,设计种属特异引物;对影响PCR扩增的因素进行分析和预试,确定PCR扩增的最佳条件;随后进行PCR扩增试验,对其产

  8. Self-organizing maps: A tool to ascertain taxonomic relatedness based on features derived from 16S rDNA sequence

    Indian Academy of Sciences (India)

    D V Raje; H J Purohit; Y P Badhe; S S Tambe; B D Kulkarni

    2010-12-01

    Exploitation of microbial wealth, of which almost 95% or more is still unexplored, is a growing need. The taxonomic placements of a new isolate based on phenotypic characteristics are now being supported by information preserved in the 16S rRNA gene. However, the analysis of 16S rDNA sequences retrieved from metagenome, by the available bioinformatics tools, is subject to limitations. In this study, the occurrences of nucleotide features in 16S rDNA sequences have been used to ascertain the taxonomic placement of organisms. The tetra- and penta-nucleotide features were extracted from the training data set of the 16S rDNA sequence, and was subjected to an artificial neural network (ANN) based tool known as self-organizing map (SOM), which helped in visualization of unsupervised classification. For selection of significant features, principal component analysis (PCA) or curvilinear component analysis (CCA) was applied. The SOM along with these techniques could discriminate the sample sequences with more than 90% accuracy, highlighting the relevance of features. To ascertain the confidence level in the developed classification approach, the test data set was specifically evaluated for Thiobacillus, with Acidiphilium, Paracocus and Starkeya, which are taxonomically reassigned. The evaluation proved the excellent generalization capability of the developed tool. The topology of genera in SOM supported the conventional chemo-biochemical classification reported in the Bergey manual.

  9. Self-organizing maps: a tool to ascertain taxonomic relatedness based on features derived from 16S rDNA sequence.

    Science.gov (United States)

    Raje, D V; Purohit, H J; Badhe, Y P; Tambe, S S; Kulkarni, B D

    2010-12-01

    Exploitation of microbial wealth, of which almost 95% or more is still unexplored, is a growing need. The taxonomic placements of a new isolate based on phenotypic characteristics are now being supported by information preserved in the 16S rRNA gene. However, the analysis of 16S rDNA sequences retrieved from metagenome, by the available bioinformatics tools, is subject to limitations. In this study, the occurrences of nucleotide features in 16S rDNA sequences have been used to ascertain the taxonomic placement of organisms. The tetra- and penta-nucleotide features were extracted from the training data set of the 16S rDNA sequence, and was subjected to an artificial neural network (ANN) based tool known as self-organizing map (SOM), which helped in visualization of unsupervised classification. For selection of significant features, principal component analysis (PCA) or curvilinear component analysis (CCA) was applied. The SOM along with these techniques could discriminate the sample sequences with more than 90% accuracy, highlighting the relevance of features. To ascertain the confidence level in the developed classification approach, the test data set was specifically evaluated for Thiobacillus, with Acidiphilium, Paracocus and Starkeya, which are taxonomically reassigned. The evaluation proved the excellent generalization capability of the developed tool. The topology of genera in SOM supported the conventional chemo-biochemical classification reported in the Bergey manual.

  10. 山羊奇异变形杆菌的分离鉴定及16SrDNA和ZapA基因的PCR-RFLP分析%Isolation and identification of Proteus mirabilis from goat and the analysis of 16S rDNA and ZapA gene by PCR-RFLP

    Institute of Scientific and Technical Information of China (English)

    王豪举; 倪莉; 杨红军; 赵俊; 徐宗可; 徐强; 徐丽敏

    2011-01-01

    Five strains of pathogen were isolated from the infected goats. Swarming behaviour assays, biochemical, animal and antibiotic resistance tests were carried out to identify its properties. Then 16S rDNA and ZapA were cloned and sequenced and both 16S rDNA and ZapA were analyzed with PCR-PFLP after digestion by specific restriction enzymes. The data showed the field strains had the swarming behaviour,which were sensitive to amikacin and for- turn,the LDs0 in mice was from 2. 500 0× 10^7 CFU/mL to 4. 445 3× 10^7CFU/mL. The DNA sequencing showed 99. 5%-99.8% similarity with Proteus mirabilis for 16S rDNA and 95. 5% for ZapA gene; the segments from PCRRFLP analysis displayed no disparity with reference strain in number and size. The results indicate that the field strains are a member of Proteus rnirabilis with no polymorphism.%从发病山羊分离到5株病原菌,对分离株进行游散行为分析,生化试验、动物试验、药敏试验及16SrDNA和ZapA基因克隆测序,选用限制性内切酶对分离株16SrDNA和ZapA基因进行PCR-RFLP分析。结果表明分离株出现游散生长现象,对丁胺卡那霉素和复达欣敏感,小鼠LD5。为2.5000×10 ^7CFU/mL-4.4453×10^7CFU/mL;分离株16SrDNA与奇异变形杆菌的同源率为99.5%~99.8%,ZapA基N与奇异变形杆菌的同源率为99.5%;PCR—RFLP分析发现,分离株酶切片段数目和大小与标准株相同。结果表明分离株是奇异变形杆菌,多态性较为单一。

  11. Sequence length variation of internal genic space of 16S rDNA-23S rDNA in biohydrogen-bacterium%产氢菌的16S -23S rDNA间隔区的长度变异性分析

    Institute of Scientific and Technical Information of China (English)

    李永峰; 郑国香; 张文启; 李建政; 胡立杰

    2005-01-01

    生物制氢细菌Rennanqilyf3的16S rRNA gene (rDNA)-23S rDNA间隔区碱基序列被测定.利用PCR扩增间隔区DNA,间隔区碱基序列存在长度多态现象.用这一长度多态现象进行产氢发酵细菌的辨认和识别.产氢发酵细菌Rennanqilyf3的16S rRNA gene (rDNA)-23S rDNA间隔区的PCR产品从1 270 到398 bp,共有5个序列.碱基数目分别为1 270、398、638、437 和 436 bp.%A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species for fermentative biohydrogen-producing bacterium. The sizes of the PCR products varied from 1 270 to 398 bp. Strain of Rennanqilyf3 were characterized as having products of 1 270,398,638, 437 and 436bp.

  12. Usefulness of 16S rDNA sequencing for the diagnosis of infective endocarditis caused by Corynebacterium diphtheriae.

    Science.gov (United States)

    Pathipati, Padmaja; Menon, Thangam; Kumar, Naveen; Francis, Thara; Sekar, Prem; Cherian, Kotturathu Mammen

    2012-08-01

    We report a rare case of infective endocarditis caused by Corynebacterium diphtheriae in an 8-year-old boy, 2 years after a right ventricular outflow tract reconstruction with a bovine Contegra valved conduit. The patient recovered well after an RV-PA conduit enblock explantation and replacement with an aortic homograft with antibiotic treatment. All bacteriological cultures of excised tissue and blood were negative. The aetiological agent was identified as C. diphtheriae subsp. gravis by 16s rDNA sequencing.

  13. Investigation of the effect of type 2 diabetes mellitus on subgingival plaque microbiota by high-throughput 16S rDNA pyrosequencing.

    Science.gov (United States)

    Zhou, Mi; Rong, Ruichen; Munro, Daniel; Zhu, Chunxia; Gao, Xiang; Zhang, Qi; Dong, Qunfeng

    2013-01-01

    Diabetes mellitus is a major risk factor for chronic periodontitis. We investigated the effects of type 2 diabetes on the subgingival plaque bacterial composition by applying culture-independent 16S rDNA sequencing to periodontal bacteria isolated from four groups of volunteers: non-diabetic subjects without periodontitis, non-diabetic subjects with periodontitis, type 2 diabetic patients without periodontitis, and type 2 diabetic patients with periodontitis. A total of 71,373 high-quality sequences were produced from the V1-V3 region of 16S rDNA genes by 454 pyrosequencing. Those 16S rDNA sequences were classified into 16 phyla, 27 classes, 48 orders, 85 families, 126 genera, and 1141 species-level OTUs. Comparing periodontally healthy samples with periodontitis samples identified 20 health-associated and 15 periodontitis-associated OTUs. In the subjects with healthy periodontium, the abundances of three genera (Prevotella, Pseudomonas, and Tannerella) and nine OTUs were significantly different between diabetic patients and their non-diabetic counterparts. In the subjects carrying periodontitis, the abundances of three phyla (Actinobacteria, Proteobacteria, and Bacteriodetes), two genera (Actinomyces and Aggregatibacter), and six OTUs were also significantly different between diabetics and non-diabetics. Our results show that type 2 diabetes mellitus could alter the bacterial composition in the subgingival plaque.

  14. Investigation of the effect of type 2 diabetes mellitus on subgingival plaque microbiota by high-throughput 16S rDNA pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Mi Zhou

    Full Text Available Diabetes mellitus is a major risk factor for chronic periodontitis. We investigated the effects of type 2 diabetes on the subgingival plaque bacterial composition by applying culture-independent 16S rDNA sequencing to periodontal bacteria isolated from four groups of volunteers: non-diabetic subjects without periodontitis, non-diabetic subjects with periodontitis, type 2 diabetic patients without periodontitis, and type 2 diabetic patients with periodontitis. A total of 71,373 high-quality sequences were produced from the V1-V3 region of 16S rDNA genes by 454 pyrosequencing. Those 16S rDNA sequences were classified into 16 phyla, 27 classes, 48 orders, 85 families, 126 genera, and 1141 species-level OTUs. Comparing periodontally healthy samples with periodontitis samples identified 20 health-associated and 15 periodontitis-associated OTUs. In the subjects with healthy periodontium, the abundances of three genera (Prevotella, Pseudomonas, and Tannerella and nine OTUs were significantly different between diabetic patients and their non-diabetic counterparts. In the subjects carrying periodontitis, the abundances of three phyla (Actinobacteria, Proteobacteria, and Bacteriodetes, two genera (Actinomyces and Aggregatibacter, and six OTUs were also significantly different between diabetics and non-diabetics. Our results show that type 2 diabetes mellitus could alter the bacterial composition in the subgingival plaque.

  15. Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.

    Science.gov (United States)

    Black, W C; Piesman, J

    1994-01-01

    Ticks are parasitiform mites that are obligate hematophagous ectoparasites of amphibians, reptiles, birds, and mammals. A phylogeny for tick families, subfamilies, and genera has been described based on morphological characters, life histories, and host associations. To test the existing phylogeny, we sequenced approximately 460 bp from the 3' end of the mitochondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick species; a mesostigmatid mite, Dermanyssus gallinae, was used as an outgroup. Phylogenies derived using distance, maximum-parsimony, or maximum-likelihood methods were congruent. The existing phylogeny was largely supported with four exceptions. In hard ticks (Ixodidae), members of Haemaphysalinae were monophyletic with the primitive Amblyomminae and members of Hyalomminae grouped within the Rhipicephalinae. In soft ticks (Argasidae), the derived phylogeny failed to support a monophyletic relationship among members of Ornithodorinae and supported placement of Argasinae as basal to the Ixodidae, suggesting that hard ticks may have originated from an Argas-like ancestor. Because most Argas species are obligate bird octoparasites, this result supports earlier suggestions that hard ticks did not evolve until the late Cretaceous. PMID:7937832

  16. The phylogeny of native and exotic scallops cultured in China based on 16S rDNA sequences

    Institute of Scientific and Technical Information of China (English)

    LIU Baozhong; DONG Bo; XIANG Jianhai; WANG Zaizhao

    2007-01-01

    Scallops of the Family Pectinidae are a valuable resource in marine industry of the world.Understanding the phylogeny of the family is important for the development of the industry. In this study,partial 16S mitochondrial rDNA genes were obtained from 8 scallop species that are commonly cultured indigenous and transplanted species in China. Phylogenetic relationships of Pectinidae were analyzed based on the 8 sequences and other 5 published ones in GenBank, representing 9 genera of the family. The molecular phylogeny trees were constructed using 3 methods with software PHYLIP. The results showe that total 13 species of scallops clustered in 4 clades. Pecten maximus joins P. jacobaeus then Amusium pleuronectes in cluster, indicating close relationship of genus Amusium with Pecten in evolution. P. yessoensis is close to Chlamysfarreri and C. islandica. No enough material was available to single out genus Patinopecten as an independent monophyletic subfamily. The position ofAdamussium colbecki indicates that it is far from genus Pecten but near to genus Chlamys in evolution.

  17. Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences.

    Science.gov (United States)

    Zhao, Ya-E; Wu, Li-Ping

    2012-09-01

    To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85 % in D. folliculorum, 98.53 % in Demodex canis, and 99.71 % in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1 %), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.

  18. The use of 16s rDNA methods in soil microbial ecology Uso de métodos 16S rDNA em ecologia microbiana do solo

    Directory of Open Access Journals (Sweden)

    Andrew Macrae

    2000-06-01

    Full Text Available New and exciting molecular methods, many using the 16S small sub-unit ribosomal nucleic acid molecule, are opening the microbial "black box" in soil. These studies have added much to our knowledge of microbial diversity in soils, and are beginning to advance our understanding of the relationship between this diversity and its function in soil processes. Over the next few years, the knowledge gained from molecular studies will, we hope, lead to improvements in sustainable land management and sustainable exploitation of soil genetic resources. As we enter the third millenium, it is appropriate to review the application of 16S rDNA methods to soil microbiology. This review examines 16S ribosomal DNA (rDNA methods and their application to soil. It mentions their limits and suggests how they may be applied in the future.Novas e excitantes técnicas moleculares muitas usando a fração 16S da subunidade menor da molécula de ácido nucleico ribossomal, estão abrindo a "caixa-preta" da microbiologia do solo. Esses estudos têm acrescentado muito ao nosso conhecimento acerca da diversidade microbiana no solo, e começam a avançar nosso entendimento sobre a relação entre essa diversidade a sua função nos processos no solo. Ao longo dos próximos anos, o conhecimento obtido a partir de técnicas moleculares irão, esperamos, levar a melhoramentos do manejo de áreas sustentáveis da exploração dos recursos genéticos do solo. Com a chegada do terceiro milênio, é apropriado revermos a aplicação das técnicas da fração 16S do rDNA em microbiologia de solo. Esta revisão examina aplicações das técnicas da fração 16S do DNA (RNA no solo, menciona seus limites e sugere como elas poderão ser usadas no futuro.

  19. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR

    Directory of Open Access Journals (Sweden)

    Wang Nian

    2009-03-01

    Full Text Available Abstract Background Citrus Huanglongbing (HLB is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. Results We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Conclusion Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

  20. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    Directory of Open Access Journals (Sweden)

    Érico Leandro da Silveira

    2006-10-01

    Full Text Available Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA and an eucalyptus arboretum (EAA. PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.Estudos sobre impacto do Eucalyptus spp. em solos brasileiros têm focalizado propriedades químicas do solo e isolamento de microrganismos de interesse. No Brasil há pouco enfoque em ecologia e diversidade microbiana, devido às limitações dos métodos tradicionais de cultivo e isolamento. A utilização de métodos moleculares no estudo da ecologia microbiana baseados na amplificação por PCR do 16S rDNA têm enriquecido o conhecimento da biodiversidade microbiana dos solos. O objetivo deste trabalho foi comparar e estimar a diversidade bacteriana de comunidades simpátricas em solos de duas áreas: uma floresta nativa (NFA e outra adjacente com arboreto de eucaliptos (EAA. Oligonucleotídeos iniciadores foram utilizados para amplificar o 16S rDNA metagenômico do solo, o qual foi

  1. 厚荚相思树根瘤菌HJ06菌株的16S rDNA全序列和nifA基因片段分析%16S rDNA and nifA Gene Sequence Analysis of Rhizobium Strain H J06 Isolated from Acacia crassicarpa

    Institute of Scientific and Technical Information of China (English)

    吕成群; 黄宝灵; 庄培亮; 武波

    2006-01-01

    对厚荚相思(Acacia crassicarpa)根瘤菌HJ06菌株的16S rDNA全序列和nifA基因片段进行了测定.结果表明,HJ06菌株在以16SrDNA序列构建的系统发育树状图中位于根瘤菌属(Rhizobium)分支中,与根瘤菌属各个种的相似性达95%以上;从HJ06菌株克隆出的585 bp nifA基因片段与Klebsiella pneumoniae的同源性达到99.3%,与Klebsiella oxytoca的NifF,NifL,NifA,NifB蛋白基因的同源性为97.8%.

  2. 16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes1

    Science.gov (United States)

    Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

    2014-01-01

    Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships. PMID:25937672

  3. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences.

    Science.gov (United States)

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen

    2012-03-23

    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  4. Analysis of the bacterial diversity in the intestine of larval Procecidochares utilis (Diptera: Trypetidae) based on 16S rDNA gene sequence%基于16S rDNA基因序列的泽兰实蝇幼虫肠道细菌多样性分析

    Institute of Scientific and Technical Information of China (English)

    张某; 杨璞; 朱家颖; 袁远; 桂富荣; 高熹; 吴国星

    2016-01-01

    [目的]探究泽兰实蝇Procecidochares utilis幼虫肠道细菌的多样性.[方法]利用Illumina HiSeq技术对泽兰实蝇幼虫肠道细菌的16S rDNA-V6变异区序列进行测序,应用USEARCH和QIIME等软件整理和统计样品序列数目和操作分类单元(operational taxonomic unit,OTU)数量,分析物种的丰度和Alpha多样性.[结果]共获得1 593 506对reads,拼接为1 579 372条tags,经过滤后得到的1 572 860条tags聚类为1 341个OTU.总共注释到13个门,4个纲,6个目,7个科,10个属和4个种.其中变形菌门(Proteobacteria)的细菌为优势菌,占99%;在属分类阶元上,沃尔巴克氏体属Wolbachia占45%,是优势属.[结论]泽兰实蝇幼虫肠道细菌多样性丰富.相关的种类和丰度信息为后期研究揭示肠道细菌介导泽兰实蝇寄主植物专化性奠定了基础.

  5. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    Science.gov (United States)

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.

  6. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

    Science.gov (United States)

    Mosher, Jennifer J; Bowman, Brett; Bernberg, Erin L; Shevchenko, Olga; Kan, Jinjun; Korlach, Jonas; Kaplan, Louis A

    2014-09-01

    Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. 16S rDNA在婴幼儿配方乳粉阪崎肠杆菌鉴定中的应用%Application of 16S rDNA in Enterobacter Sakazakii Identiifcation for Infant Formula Milk Powder

    Institute of Scientific and Technical Information of China (English)

    林玉宙

    2015-01-01

    选取近几年从婴幼儿配方乳粉生产过程环境及产品中收集、经国标GB/T 4789.40-2010检验和API20E鉴定为阪崎肠杆菌阳性的菌株16株,利用MicroSEQ ID微生物鉴定系统进行16S rDNA基因测序分析,构建系统发育树,鉴定种属。结果显示,这16份经API20E鉴定为阪崎肠杆菌阳性的菌株,经16S rDNA基因测序证实,其中2份为梨形肠杆菌,1份为克氏柠檬酸杆菌,其余13份样品均为阪崎肠杆菌,且与数据库中标准菌株的同源性达到99.5%以上。16S rDNA基因测序方法在婴幼儿配方乳粉企业进行阪崎肠杆菌鉴定和溯源方面具有广阔的前景。%Sixteen Enterobacter sakazaki strains col ected from the environment and products during infant formula milk powder production process and identiifed to be positive by GB/T 4789.40-2010 as wel as API20E were analyzed by 16S rDNA gene se-quencing using MicroSEQ ID microbial identiifcation system,and the system development tree was built to identify the species. The results showed that two of 16 Enterobacter sakazaki strains were Enterobacter pyrinus,and one was Citrobacter koseri,and the other thirteen were Enterobacter sakazaki and the homology with standard Enterobacter sakazaki strain in the database was more than 99.5%. 16S rDNA gene sequencing method has the broad prospect in the identiifcation and traceability of Enterobacter saka-zaki for infant formula enterprises.

  8. Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures.

    Science.gov (United States)

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga-Flavobacterium-Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities.

  9. Diversity and phylogenetic analysis of endosymbiotic bacteria from field caught Bemisia tabaci from different locations of North India based on 16S rDNA library screening.

    Science.gov (United States)

    Singh, Shalini Thakur; Priya, Natarajan Gayatri; Kumar, Jitendra; Rana, Vipin Singh; Ellango, R; Joshi, Adita; Priyadarshini, Garima; Asokan, R; Rajagopal, Raman

    2012-03-01

    Bemisia tabaci is the major vector pest of agricultural crops all over the world. In this study we report the different bacterial endosymbionts associated with B. tabaci sampled from 14 different locations in North India. Using 16S rDNA clone library sequences we were able to identify Portiera, the primary endosymbiont of B. tabaci, and other secondary endosymbionts like Cardinium, Wolbachia, Rickettsia and Arsenophonus. Along with these we also detected Bacillus, Enterobacter, Paracoccus and Acinetobacter. These secondary endosymbionts were not uniformly distributed in all the locations. Phylogenetic analysis of 16S rDNA sequences of Cardinium, Wolbachia, Rickettsia and Arsenophonus showed that each of these bacteria form a separate cluster when compared to their respective counterparts from other parts of the world. MtCO1 gene based phylogenetic analysis showed the presence of Asia I and Asia II genetic groups of B. tabaci in N. India. The multiple correspondence analyses showed no correlation between the host genetic group and the endosymbiont diversity. These results suggest that the bacterial endosymbiont diversity of B. tabaci is much larger and complex than previously perceived and probably N. Indian strains of the bacterial symbionts could have evolved from some other ancestor.

  10. Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis (Hemiptera: Aleyrodidae) adults using 16S rDNA amplicon pyrosequencing and FISH.

    Science.gov (United States)

    Pandey, Neeti; Rajagopal, Raman

    2016-10-01

    Dialeurolonga malleswaramensis Sundararaj (Hemiptera: Aleyrodidae) is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene (mtCO1) helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswaramensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization (FISH) in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.

  11. 基于16S rDNA序列和RFLP分析的病鳗分离菌株鉴定%Identification of Bacteria Isolated from Diseased Eels Using 16S rDNA PCR / RFLP Analysis

    Institute of Scientific and Technical Information of China (English)

    郭松林; 关瑞章; 冯建军; 杨求华

    2012-01-01

    Using 16S rDNA gene sequences and restriction fragment length polymorphism (RFLP) a- nalysis, the study initially identified 30 strains of bacteria isolated from European eel (Anguilla anguilla) , Japanese eel (Anguilla japonica) and American eel (Anguilla rostrata) according to the homology compari- son with bacterial 16S rDNA gene sequence which had been submitted into the C, eneBank database. The re- sults showed that the 30 bacteria could be broadly divided into 7 genera, including Aeromonas sp. , Pseudo- monas sp. , P/es/omonas sp. , Klebsh:lla sp. , Citrobacter sp. , Acinetobacter sp. and Enterobacter sp. Re- sults of the study indicated some of the isolates could be suspected as pathogenic strains which caused diseases to eels.%结合16S rDNA基因序列和限制性片段长度多态性(RFLP)的分析方法,通过与GenBank库中已递交的细菌16S rDNA基因序列进行同源性比较,对分离自发病鳗鲡(欧洲鳗鲡,日本鳗鲡和美洲鳗鲡)的30株细菌进行初步鉴定和分类.结果表明,这些细菌可大致分为气单胞菌属(Aeromonas sp.)、假单胞菌属(Pseudomonas sp.)、邻单胞菌属(Plesiomonas sp.)、克雷伯氏菌属(Klebsiella sp.)、柠檬酸杆菌属(Citrobacter sp.)、不动杆菌属(Acinetobacter sp.)和肠杆菌属(Enterobacter sp.)等7个菌属.分析认为,部分分离菌株可能是引起鳗鲡病害的疑似致病性菌株.

  12. Uptake of copper ion by activated sludge and its bacterial community variation analyzed by 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The effect and uptake of copper ion on SBR(sequence batch reactor) biological treatment system was studied in this paper. Special nutrient and powder activated carbon(PAC) additive were tested as uptake stimulation technique. Results showed that copper ion had higher effect on unacclimated activated sludge system than on acclimated one. The special nutrient adding could enhance the uptake of copper significantly, while PAC adding could improve the sludge settling and decrease the turbidity of effluent. The variation of bacterial community analyzed by 16S rDNA method showed the acclimation of copper could increase copper resistance species, and excess accumulation could cause some species diminish. It was confirmed that acclimation could improve the resistance and uptake ability of microorganism to heavy metal.

  13. Isolation of culturable endophytic bacteria from Moso bamboo (Phyllostachys edulis and 16S rDNA diversity analysis

    Directory of Open Access Journals (Sweden)

    Yuan Zong-Sheng

    2015-01-01

    Full Text Available We analyzed culturable endophytic bacteria from Moso bamboo (Phyllostachys edulis using traditional bacterial isolation and culture methods and then studied the colony characteristics and diversity with a 16S rDNA sequence analysis. We isolated 82 endophytic bacteria strains belonging to 47 species in 26 genera from the root, rhizome, stem and leaves of Moso bamboo species from populations on Wuyi Mountain, and in the Jiangle and Changting regions. There were significant differences in the composition of the culturable endophytic bacteria isolated from the different areas and from different tissues. The dominant bacteria strains from the Wuyi Mountain samples were Arthrobacter, Staphylococcus, Bacillus and Enterobacter, while the dominant bacteria from the Jiangle samples were Bacillus, Staphylococcus and Curtobacterium, and the dominant bacteria in the Changting samples were Alcaligenes, Pseudomonas, Staphylococcus and Bacillus. Our results demonstrate the abundant diversity of endophytic bacteria in Moso bamboo.

  14. Molecular identification of four phenotypes of human Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA.

    Science.gov (United States)

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Classification of Demodex mites has long depended on hosts and morphological characteristics. However, the fact that two species coexist in the same host and phenotype is easily influenced by environment causes difficulty and indeterminacy in traditional classification. Genotype, which directly reflects the molecular structure characteristics, is relatively stable. In this study, species identification of four phenotypes of human Demodex mites was conducted. Mites were morphologically classified into four phenotypes: long- and short-bodied Demodex folliculorum with finger-like terminus and Demodex brevis with finger- or cone-like terminus. The mitochondrial 16S ribosomal DNA (rDNA) fragment of individual mite was amplified, cloned, sequenced, and aligned. Sequence divergences, genetic distances, transition/transversion rates, and phylogenetic trees were analyzed. The results demonstrated that the 16S rDNA sequence of three phenotypes with finger-like terminus was 337 bp, and that of phenotype with cone-like terminus was 342 bp. The divergences, genetic distances, and transition/transversion rates among the three phenotypes with finger-like terminus were 0.0-2.7%, 0.000-0.029, and 5.0-7/0 (5/1-7/0), respectively, indicating an intraspecific variation. Yet, those between these three phenotypes and the one with cone-like terminus were 21.6-22.8%, 2.510-2.589, and 0.47-0.59 (22/47-27/46), respectively, suggesting an interspecific variation. The five phylogenetic trees showed that the three phenotypes with finger-like terminus clustered into one branch, while the phenotype with cone-like terminus clustered into another. In conclusion, terminus is a major morphological characteristic for the identification of human Demodex species. The three phenotypes with finger-like terminus belong to D. folliculorum, while the phenotype with cone-like terminus belongs to D. brevis. Molecular identification can verify and replenish morphological identification.

  15. TURKEY FECAL MICROBIAL COMMUNITY STRUCTURE AND ECOLOGICAL FUNCTIONS REVEALED BY 16S RDNA AND METAGENOME SEQUENCES

    Science.gov (United States)

    Turkey feces are an important source of fecal waste in the United States. With the exception of isolated studies on bacterial pathogens, little is known about the type of bacteria inhabiting the turkey gut. In order to understand the microbial diversity and functional genes assoc...

  16. Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis.

    Directory of Open Access Journals (Sweden)

    Robert E Tyx

    Full Text Available The bacterial communities present in smokeless tobacco (ST products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae. The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

  17. Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis.

    Science.gov (United States)

    Tyx, Robert E; Stanfill, Stephen B; Keong, Lisa M; Rivera, Angel J; Satten, Glen A; Watson, Clifford H

    2016-01-01

    The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

  18. Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods.

    Science.gov (United States)

    Takahashi, Hajime; Kimura, Bon; Tanaka, Yuichiro; Mori, Mayumi; Yokoi, Asami; Fujii, Tateo

    2008-04-01

    The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.

  19. Distribution and 16S rDNA sequences of Argas monachus (Acari: Argasidae), a soft tick parasite of Myiopsitta monachus (Aves: Psittacidae).

    Science.gov (United States)

    Mastropaolo, Mariano; Turienzo, Paola; Di Iorio, Osvaldo; Nava, Santiago; Venzal, José M; Guglielmone, Alberto A; Mangold, Atilio J

    2011-11-01

    Specimens of Argas monachus Keirans et al. were collected from Myiopsitta monachus nests in 42 localities in Argentina and Paraguay from 2006 to 2010. A list of localities where this tick has been found is presented. 16S rDNA sequences of specimens of A. monachus from different localities were compared to confirm whether they belong to the same specific taxon. Argas monachus is present in the phytogeographic provinces of Chaco, Espinal, and Monte, but not in the Pampa (all from de Chaco Domain) where the host is well distributed. No differences were found among 16S rDNA sequences of geographically distant specimens.

  20. 棘阿米巴土壤分离株CB/S1内共生细菌的16SrDNA序列分析%Sequence Analysis of 16S rDNA Gene of Endosymbiont of Acanthamoeba sp.CB/S1 Isolated from Soil

    Institute of Scientific and Technical Information of China (English)

    玄英花; 崔春权; 郑善子

    2011-01-01

    The endosymbiont of Acanthamoeba sp. CB/S1 was identified by orcein-carmine staining and 16S rDNA sequence analysis. The endosymbiont bacteria were rod-shaped and darkly stained, and irregularly localized within the cytoplasm. The length of the 16S rDNA was 1534 bp and its DNA sequence was closely related to those of Candidatus A moebophilus asiaticus and A canthamoeba sp. KA/E21 with 98% homology. Phylogenetic analysis showed that the endosymbiont of CB/S1,the endosymbiont of KA/E21, Candidatus Amoebophilus asiaticus, the endosymbiont of Ixodes scapularis , and the endosymbiont of Encarsia pergandiella constitute a monophyletic lineage in phylogenetic tree.%用地衣红-卡红染色进行共生菌的形态观察,鉴定棘阿米巴CB/S1内共生细菌.克隆内共生细菌的16S rDNA基因,进行基因序列分析.结果 表明,经地衣红-卡红染色棘阿米巴CB/S1内共生细菌呈黑色和棒状,在胞质内不规则分布.棘阿米巴CB/S1内共生细菌的16S rDNA基因长1 534 bp,与类亚洲嗜阿米巴杆菌(Candidatus Amoebophilus asiaticus 5a2)和韩国棘阿米巴分离株 KA/E21内共生细菌的16S rDNA基因的同源性均为98%.进化树分析表明,棘阿米巴CB/S1内共生细菌与韩国棘阿米巴KA/E21内共生细菌、类亚洲嗜阿米巴杆菌、黑脚硬蜱内共生细菌和伯恩蚜小蜂内共生细菌等细菌构成单系.

  1. 16S rDNA sequence as measure for identification of bacterial isolates in bulk%16S rDNA序列在大批量细菌分离物分子鉴定中的应用研究

    Institute of Scientific and Technical Information of China (English)

    陈源源; 沈微; 樊游; 陈献忠; Suren Singh; 石贵阳; 王正祥

    2013-01-01

    The feasibility of 16S rDNA sequence-based analysis for systemic classification of bacterial isolates was extensively evaluated. The 16S rDNA sequences of 3 726 bacterial strains collected in CICIM-CU were amplified by PCR and sequenced. The sequencing results were compared to reference data available at the GenBank database by using BLAST. Most of bacterial isolates (82.5% ) were assigned to species level successfully. 653 strains (17.5% ) were only could be assigned to genus level. Some closely-related species belonged to genus Bacillus and Geobacillus had highly similar 16S rDNA sequences, making 16S rDNA sequence analysis-based identification problematic. This study showed that 16S rDNA sequences were sufficiently sensitive for identifying most of bacterial isolates. It could be powerfully and ordinarily used as first-round molecular marker to systemic identification of bacterial isolates in bulk.%利用16S rDNA序列同源性分析法对保藏于中国高校工业微生物资源与信息中心(CICIM-CU)的3 726株细菌分离物进行了分子鉴定.实验结果显示:其中3 073株细菌分离物可被成功鉴定至种一级水平,分属于210个种,45个属,占整个细菌分离物的82.5%;其余653株细菌分离物可鉴定到属一级,占整个细菌分离物的17.5%.研究中也发现,16S rDNA序列在芽孢杆菌属(Bacillus)和土芽孢杆菌属(eobacillus)等少数菌属中的部分种间鉴定的敏感性不高.上述结果表明16S rDNA序列在大多数细菌种属鉴定中具有较高的特异性和敏感性,可以作为第一轮分子标识用于大批量细菌分离物的鉴定.

  2. Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

    Directory of Open Access Journals (Sweden)

    Ahmed Marroki

    2011-03-01

    Full Text Available Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21 and one strain of L. rhamnosus (LbMF25 have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.

  3. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

    Science.gov (United States)

    Armougom, F; Bittar, F; Stremler, N; Rolain, J-M; Robert, C; Dubus, J-C; Sarles, J; Raoult, D; La Scola, B

    2009-09-01

    Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

  4. Romanian cyprinids phylogeny based on 16S ARN mitochondrial genes

    OpenAIRE

    Luca C.; Kevorkian S.; Elvira M.; Dinischiotu A.; Costache M.

    2007-01-01

    The vertebrate mitochondrial genome has been an important model system for studying molecular evolution, organism phylogeny, and genome structure. Phylogenetic relatioships were inferred from analysis of 570 base pairs (bp) of mithocondrial DNA (mtDNA), representing a conserved region of 16S rRNA. We sequenced 13 cyprinids species and one putative outgroup (Misgurnus fossilis) from Romania. Based upon nucleotide sequence comparisons of cyprinid mitochondrial 16SRNA genes, we established the p...

  5. Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing.

    Science.gov (United States)

    Chen, Ya-Bing; Lan, Dao-Liang; Tang, Cheng; Yang, Xiao-Nong; Li, Jian

    2015-01-01

    To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

  6. Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

    Science.gov (United States)

    Verma, Subhash Chandra; Chowdhury, Soumitra Paul; Tripathi, Anil Kumar

    2004-05-01

    Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

  7. 蜡状芽胞杆菌群16S rDNA分析%Analysis of 16S rDNA in the Bacillus cereus Group

    Institute of Scientific and Technical Information of China (English)

    邹寰; 喻子牛; 孙明

    2008-01-01

    蜡状芽胞杆菌群主要包括炭疽芽胞杆菌(Bacillus anthracis)、蜡状芽胞杆菌(Bacillus cereus)、苏云金芽胞杆菌(Bacillus thuringiensis).GenBank已有这个群的8个菌株完成了全基因组序列测定.对这8株蜡状芽胞杆菌群菌株中98条16S rDNA的序列进行相互BLAST比较,发现在同一基因组内各个16S rDNA拷贝全局相似度最低为96.47%,在不同基因组间16S rDNA局部片段比对最小相似度达到99.72%,对应片段长度也有1417 bp.这点充分说明,该群的细菌完全共用同一种16S rDNA,根据16S rDNA给细菌分类的特点,它们应该属于同一个种.在亲缘关系上,枯草芽胞杆菌离蜡状芽胞杆菌群最近.

  8. 海南温泉嗜热菌的16S rDNA分析%Hainan Hot Spring Thermophiles' 16S rDNA Analysis

    Institute of Scientific and Technical Information of China (English)

    吴红萍; 孟甜; 张飞官; 陈永安; 李文芳; 王丙乾; 王锐萍

    2013-01-01

    目的:确定24株海南温泉嗜热菌菌株的分类地位.方法:Blastn分析菌株16S rDNA序列同源性;邻接法构建菌株16S rDNA序列系统发育进化树并分析菌株的进化位置;Clustax比对分析菌株的相似度和进化距离.结果:菌株LY5和LY4的16S rDNA序列与Geobacillus pallidus strain B1,partial sequence(GenBank:HM030740.1)的16SrDNA序列同源性分别为98%和97%,其他菌株的16S rDNA序列与Geobacillus subterraneus,strain R-35641(GenBank:FN428689.1)的16S rDNA序列的同源性均大于96%.Clustax比对分析表明26株菌16S rDNA序列前段(1~70bp)、中段(70bp~1420bp)、后段(1420~1484bp)的相似度分别为40%、100%和60%,进化距离分析表明菌株GT7、LY4和LY5与其他菌株进化距离较远,其余菌株之间进化距离差异不明显.综上所述,初步将24株温泉嗜热菌鉴定为土芽孢杆菌属(Geobacillus sp.).结论:16S rDNA序列分析可用于温泉嗜热菌的鉴定.

  9. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene

    Directory of Open Access Journals (Sweden)

    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S. Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more clinical and reference strains.

  10. Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene.

    Directory of Open Access Journals (Sweden)

    Nelson Enrique Arenas

    2009-12-01

    Full Text Available Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S.Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical and reference strains.

  11. A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library.

    Science.gov (United States)

    Manichanh, Chaysavanh; Chapple, Charles E; Frangeul, Lionel; Gloux, Karine; Guigo, Roderic; Dore, Joel

    2008-09-01

    The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10,010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper.

  12. Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

    Directory of Open Access Journals (Sweden)

    Fouts Derrick E

    2012-08-01

    Full Text Available Abstract Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI. Asymptomatic bacteriuria (ABU represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein

  13. Bacterial diversity in soil samples from two uranium waste piles as determined by rep-APD, RISA and 16S rDNA retrieval.

    Science.gov (United States)

    Selenska-Pobell, S; Kampf, G; Hemming, K; Radeva, G; Satchanska, G

    2001-06-01

    The bacterial diversity in two uranium waste piles was studied. Total DNA was recovered from a large number of soil samples collected from different sites and depths in the piles using two procedures for direct lysis. Significant differences in the bacterial composition of the samples were revealed by the use of rep-APD, RISA and 16S ARDREA. The 16S rDNA analyses showed that the uranium wastes were dominated by Acidithiobacillusferrooxidans and by several Pseudomonas species classified in the gamma-subdivision of the Proteobacteria. The three kinds of A. ferrooxidans 16S and IGS rDNA specific fragments that were found corresponded to the three phylogenetic groups recognised in this species. This microdiversity probably reflects the genetic adaptation of the uranium waste strains to different concentrations of heavy metals.

  14. High throughput 16S rRNA gene amplicon sequencing

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Jørgensen, Mads Koustrup

    S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution. In this study we show how 16S r...... to the presence of filamentous microorganisms was monitored weekly over 4 months. Microthrix was identified as a causative filament and suitable control measures were introduced. The level of Microthrix was reduced after 1-2 months but a number of other filamentous species were still present, with most of them...

  15. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

    Directory of Open Access Journals (Sweden)

    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  16. Phylogeny of the cuttlefishes (Mollusca:Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

    Institute of Scientific and Technical Information of China (English)

    LIN Xiangzhi; ZHENG Xiaodong; XIAO Shu; WANG Rucai

    2004-01-01

    To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

  17. Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

    Science.gov (United States)

    Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

    2004-06-15

    The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

  18. [Molecular identification and detection of moon jellyfish (Aurelia sp.) based on partial sequencing of mitochondrial 16S rDNA and COI].

    Science.gov (United States)

    Wang, Jian-Yan; Zhen, Yu; Wang, Guo-shan; Mi, Tie-Zhu; Yu, Zhi-gang

    2013-03-01

    Taking the moon jellyfish Aurelia sp. commonly found in our coastal sea areas as test object, its genome DNA was extracted, the partial sequences of mt-16S rDNA (650 bp) and mt-COI (709 bp) were PCR-amplified, and, after purification, cloning, and sequencing, the sequences obtained were BLASTn-analyzed. The sequences of greater difference with those of the other jellyfish were chosen, and eight specific primers for the mt-16S rDNA and mt-COI of Aurelia sp. were designed, respectively. The specificity test indicated that the primer AS3 for the mt-16S rDNA and the primer AC3 for the mt-COI were excellent in rapidly detecting the target jellyfish from Rhopilema esculentum, Nemopilema nomurai, Cyanea nozakii, Acromitus sp., and Aurelia sp., and thus, the techniques for the molecular identification and detection of moon jellyfish were preliminarily established, which could get rid of the limitations in classical morphological identification of Aurelia sp. , being able to find the Aurelia sp. in the samples more quickly and accurately.

  19. Evaluation of direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water.

    Science.gov (United States)

    Hansen, Trine; Skånseng, Beate; Hoorfar, Jeffrey; Löfström, Charlotta

    2013-09-01

    Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations.

  20. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    KAUST Repository

    Qian, Peiyuan

    2010-07-29

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea. © 2011 International Society for Microbial Ecology All rights reserved.

  1. 不同刺槐种源根瘤菌16S rDNA的PCR-RFLP分析%PCR-RFLP Analysis of 16S rDNA of Rhizobia Isolated from Different Provenances of Robinia pseudoacacia

    Institute of Scientific and Technical Information of China (English)

    张泽坤; 韩骞; 杨敏生; 王进茂

    2012-01-01

    为了研究不同刺槐种源根瘤茵的遗传多样性,采用4种限制性内切酶对分离自保定和高邑的38株不同刺槐种源根瘤茵进行16S rDNA PCR-RFLP多态性分析,共产生26种16SrDNA遗传图谱类型.UPGMA聚类分析结果显示,所有供试菌株分为4大类,第3类又分为3个亚类.16S rDNA全序列分析表明,代表菌株GY-2与S.morelense LMG20571序列相似性达到99.6%,在系统发育分类地位上属于Sinorhizobium.刺槐根瘤菌遗传多样性丰富,遗传差异受地理环境影响较大,与寄主种源相关性不明显.%A study was performed to analyze the genetic diversity of rhizobia isolated from different provenances of Robinia pseud-oacacia. PCR-RFLP polymorphism analysis of 16S rDNA of 38 rhizobia grown in Baoding and Gaoyi was made with four restriction endonucleases. A total of 26 geneticmap patterns were generated from the tested strains. UPGMA cluster analysis indicates that all strains can be clustered into four groups, and the third group into three subgroups. 16S rDNA sequence analysis indicates that the representative strain GY-2 has a similarity level of 99. 6% with Sinorhizobium morelense LMG20571, and belongs to Sinorhizobium in the phylogenetic classification status. Results show that the rhizobia isolated from R. pseudoacacia reveal a high level of genetic diversity, and genetic differences are influenced by geographical environment, but its correlation with host provenance is not obvious.

  2. Identification of Enterobacter sakazakii from closely related species: The use of Artificial Neural Networks in the analysis of biochemical and 16S rDNA data

    Directory of Open Access Journals (Sweden)

    Waddington Michael

    2006-03-01

    Full Text Available Abstract Background Enterobacter sakazakii is an emergent pathogen associated with ingestion of infant formula and accurate identification is important in both industrial and clinical settings. Bacterial species can be difficult to accurately characterise from complex biochemical datasets and computer algorithms can potentially simplify the process. Results Artificial Neural Networks were applied to biochemical and 16S rDNA data derived from 282 strains of Enterobacteriaceae, including 189 E. sakazakii isolates, in order to identify key characteristics which could improve the identification of E. sakazakii. The models developed resulted in a predictive performance for blind (validation data of 99.3 % correct discrimination between E. sakazakii and closely related species for both phenotypic and genotypic data. Three main regions of the partial rDNA sequence were found to be key in discriminating the species. Comparison between E. sakazakii and other strains also constitutively positive for expression of the enzyme α-glucosidase resulted in a predictive performance of 98.7 % for 16S rDNA sequence data and 100% for phenotypic data. Conclusion The computationally based methods developed here show a remarkable ability in reducing data dimensionality and complexity, in order to eliminate noise from the system in order to facilitate the speed and reliability of a potential strain identification system. Furthermore, the approaches described are also able to provide valuable information regarding the population structure and distribution of individual species thus providing the foundations for novel assays and diagnostic tests for rapid identification of pathogens.

  3. Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus%2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

    Institute of Scientific and Technical Information of China (English)

    邓先余; 陈晓艳; 王智学; 欧普; 何建国

    2006-01-01

    根据细菌的16S rDNA 3'端和23S rDNA 5'端的高度保守区设计引物,PCR扩增了2株创伤弧菌(Vibrio vulnificus)的16S-23S rDNA间区(Intergenic spacer,IGS),克隆到pGEM-T载体上,测序.用BLAST和DNAstar软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析.结果表明,2株创伤弧菌共测出9条16S-23S rDNA间区序列,其中ZSU006测出5条,间区类型分别为:IGSGLAV、IGSGLV、IGSIA、IGSA和IGSG.其中IGSGLAV最大,包含tRNAGlu、tRNALys、tRNAAla和tRNAVal基因;IGSGLV包含tRNAGlu、tRNALys和tRNAVal基因;IGSIn,则包含tRNAIle和tRNAAla基因;IGSG仅包含tRNAGlu基因;而IGSA仅包含tRNAAla基因.菌株CG021测出的16S-23S rDNAIGS序列有4条,除缺少IGSA外,其余的IGS类型均与ZSU006的相同.与GenBank内的创伤弧菌ATCC27562的IGS序列比较,发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性.16S-23S rDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础.%According to the conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic16S-23S rDNA intergenic spacers, namely, IGSGLAV, IGSGLv, IGSIA, IGSG and IGSA; while the strain CG021 has the same types of IGSs except lacking IGSA. Among these five IGS types, IGSGLAV is the biggest type, including the gene cluster of tRNAGlu - tRNALys - tRNAAla - tRNAVal; IGSGLV includes that of tRNAGlu-tRNALys-tRNAVal; IGSAG, tRNAAla-tRNAGlu; IGSIA, tRNAIle -tRNAAla; IGSG, tRNAGlu and IGSA, tRNAAla. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V

  4. Detection of XIIA phytoplasma group on cultivar Župljanka in Župa vineyard region by RFLP analysis of 16s rDNA sequences

    Directory of Open Access Journals (Sweden)

    Jošić Dragana

    2010-01-01

    Full Text Available 'Bois noir' (BN is an important grapevine disease associated with phytoplasmas belonging to ribosomal subgroup 16SrXII-A. Phytoplasmas cause diseases in several hundred plant species. The number of infected cultivars is growing each year and it is important to follow the spreading of the phytoplasma in the different regions and identify which strains are present in specific regions on specific cultivars. Phytoplasmas are identified and classified based on direct sequencing of phytoplasma 16S rDNA or the 16S to 23S intergenic spacer region, but this approach is not always practical when a large number of unknown phytoplasmas is to be analyzed. Classification by RFLP analysis has provided a simple and rapid method that can be used to differentiate and identify a large number of unclarified phytoplasmas. Our objective was to investigate presence of phytoplasmas of 16SrXII-A group (Stolbur in Zupa vineyard region. Detection was based on RFLP analysis of 16s rDNA sequences using four restriction enzymes: Tru1I, AluI, KpnI and TaqI. We identified phytoplasmas of XIIA group on two of three investigated cultivars (Zupljanka and Frankovka, but not on Plovdina in the Zupa vineyard regions (Gornje Rataje and Tules locality. This is the first report of Stolbur phytoplasma on cv. Zupljanka in Zupa region.

  5. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    Science.gov (United States)

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

  6. Application of bacterial 16S rDNA amplification and sequencing in the classification and identification of bacteria%16S rDNA扩增及测序在细菌鉴定与分类中的应用

    Institute of Scientific and Technical Information of China (English)

    朱诗应; 戚中田

    2013-01-01

    Bacterial 16S rDNA amplification and sequencing is a new tool which has been widely used to identify bacterial species and perform taxonomic studies . The application of this technology for identification of uncultivable bacteria , differentiating species with high DNA sequence similarity and discovering novel bacterial genus and species are introduced in this paper . Future perspective of the method in clinical microbiology laboratories is also discussed .%16S rDNA扩增及测序技术在细菌的鉴定与分类研究中发挥着越来越重要的作用.本文就16S rDNA结构、可变区和保守区部分序列或全序列在临床上细菌鉴定和新细菌识别等方面的研究进展进行综述,并对其在临床实验室中的应用进行展望.

  7. Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori

    NARCIS (Netherlands)

    M.M. Gerrits (Monique); M. Berning; A.H.M. van Vliet (Arnoud); E.J. Kuipers (Ernst); J.G. Kusters (Johannes)

    2003-01-01

    textabstractThe triple-base-pair 16S rDNA mutation AGA(926-928)-->TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence o

  8. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

    Directory of Open Access Journals (Sweden)

    Aboubaker M. Garbaj

    2016-11-01

    Full Text Available Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey that include 3 isolates from cow’s milk (11%, 3 isolates from she-camel’s milk (11%, two isolates from goat’s milk (7.4% and 7 isolates from fermented raw milk samples (26%, isolates from fresh locally made soft cheeses (Maasora and Ricotta were 9 (33% and 3 (11%, respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

  9. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA.

    Science.gov (United States)

    Garbaj, Aboubaker M; Awad, Enas M; Azwai, Salah M; Abolghait, Said K; Naas, Hesham T; Moawad, Ashraf A; Gammoudi, Fatim T; Barbieri, Ilaria; Eldaghayes, Ibrahim M

    2016-11-01

    The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow's milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow's milk (11%), 3 isolates from she-camel's milk (11%), two isolates from goat's milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

  10. Amblyomma aureolatum (Pallas, 1772) and Amblyomma ovale Koch, 1844 (Acari: Ixodidae): hosts, distribution and 16S rDNA sequences.

    Science.gov (United States)

    Guglielmone, A A; Estrada-Peña, A; Mangold, A J; Barros-Battesti, D M; Labruna, M B; Martins, J R; Venzal, J M; Arzua, M; Keirans, J E

    2003-05-01

    DNA sequences of Amblyomma aureolatum (Pallas, 1772) and Amblyomma ovale Koch, 1844 were obtained to determine genetic differences between these tick species. Collections of these species are discussed in relation to distribution and hosts. Seven ticks collections (four from Brazil, one from Argentina, one from Uruguay and one from USA) house a total of 1272 A. aureolatum (224 males, 251 females, 223 nymphs and 574 larvae) and 1164 A. ovale (535 males, 556 females, 66 nymphs and 7 larvae). The length of the sequenced mitochondrial 16S rRNA gene fragment for A. aureolatum was 370bp and for A. ovale was 373bp. The DNA sequence analysis showed a 13.1% difference between the two species. Apart from one male A. ovale found on a toad, all adult ticks were found on mammals. The majority of adult specimens of both tick species were removed from Carnivora (96.1 and 84.3% of A. aureolatum and A. ovale, respectively), especially from dogs (53.1% of A. aureolatum, and 46.4% of A. ovale). Collections on wild Canidae were higher for A. aureolatum (23.3%) than for A. ovale (7.1%). On the other hand, collections of A. ovale adults on wild Felidae were higher (18.3%) than findings of A. aureolatum (9.2%). The contribution of other mammalian orders as hosts for adults of A. aureolatum and A. ovale was irrelevant, with the exception of Perissodactyla because Tapiridae contributed with 13.0% of the total number of A. ovale adults. Adults of both tick species have been found occasionally on domestic hosts (apart of the dog) and humans. Most immature stages of A. aureolatum were found on Passeriformes birds, while rodents and carnivores were the most common hosts for nymphs and larvae of A. ovale. A. aureolatum has been found restricted to the Neotropical region, covering the eastern area of South America from Uruguay to Surinam, including northeastern Argentina, eastern Paraguay, southeastern Brazil and French Guiana. A. ovale showed a distribution that covers the Neotropical region

  11. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

    OpenAIRE

    Aboubaker M. Garbaj; Enas M. Awad; Salah M. Azwai; Said K. Abolghait; Naas, Hesham T.; Moawad, Ashraf A.; Fatim T. Gammoudi; Ilaria Barbieri; Ibrahim M. Eldaghayes

    2016-01-01

    Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice c...

  12. Analysis of 16S rDNA Sequence and DNA朌NA Hybridization of Rhizobia Isolated from Indigofera sp.%木蓝根瘤菌的16S rDNA全序列分析及DNA朌NA杂交

    Institute of Scientific and Technical Information of China (English)

    韦革宏; 陈文新; 朱铭莪

    2000-01-01

    通过数值分类、SDS-全细胞蛋白电泳分析,对分离自西北黄土高原地区的木蓝根瘤菌进行了研究,获得了1个新类群。在此基础上,进行了中心菌株SHL042的16S rDNA全序列分析,得到系统发育树状图。SHL042与R.tropici A、R.tropici B、R. leguminosarum、R. etli、R. hananesis、R. mongolense和R. gallicum构成一个发育分支,其与这些种模式菌株16S rDNA全序列的相似性分别为95.4%、95.5%、96.3%、95.8%、96.3%、97.9%和97.7%,均大于95%,应属于同一个属。新类群群内DNA同源性大于80%,而中心菌株SHL042与分支内各已知种的DNA同源性小于50%,表明SHL042代表1个新的根瘤菌菌种。%Based on the previous studies on numerical taxonomy and SDS-PAGE of whole-cell protein, the rhizobia strains isolated from Indigofera sp. in loess plateau area of North-west China constituted a new cluster, the 16S rDNA sequence of representative strain SHL042 was tested, and the phylogenetic tree was produced. In this tree, the strain SHL042, R. tropici A, R. tropici B, R. leguminosarum, R. etli, R.hananesis, R. mongolense and R. gallicum constituted a branch of phylogenetic. Within this branch, the similarity values of 16S rDNA sequence were 95.4%, 95.5%,96.3%,95.8%,96.3%,97.9% and 97.7% respectively. The values were more than 95%. This indicated that these species should belong to the same genus. The values of DNA homology in the new cluster were all more than 80%, but the values between SHL042 and the strains of these species were less than 50%. Thus, the strain SH714 represented a new rhizobia species.

  13. Predictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality

    OpenAIRE

    Delhalle, Laurent; Ellouze, Mariem; Taminiau, Bernard; Korsak Koulagenko, Nicolas; Nezer, Carine; Daube,Georges

    2013-01-01

    OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These techniques are simple to implement but may not be relevant to understand the modifications of the microbial ecology which occur in the food product in response to different changes in the environmental conditions. Metagenomic analysis targeted on 16S ribosomal DNA can bring about a solution to t...

  14. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    Science.gov (United States)

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  15. 16S rDNA RFLP Analysis of Rhizobia Isolated from Medicago lupulina in Northwestern China%西北地区天蓝苜蓿根瘤菌16S rDNA RFLP分析

    Institute of Scientific and Technical Information of China (English)

    冯春生; 郭军康; 位秀丽; 李香香; 韦革宏

    2008-01-01

    利用RFLP和序列测定方法,对分离自西北地区的67株天蓝苜蓿根瘤菌16S rDNA进行了分析研究.结果表明:所有供试菌株分别归属于中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobacterium).以CCNWNX0042-2为代表的大部分天蓝苜蓿根瘤菌属于草木樨中华根瘤菌(Sinorhizobium meliloti),其余菌株在分群上表现出了较为明显的地域特征.

  16. Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; YANG Guanpin; WANG Hualei; CHEN Jixiang; SHI Xianming; ZOU Guiwei; WEI Qiwei; SUN Xiuqin

    2006-01-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  17. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Science.gov (United States)

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  18. Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Dinka eMandakovic

    2016-05-01

    Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  19. Marine sponge Craniella austrialiensis-associated bacterial diversity revelation based on 16S rDNA library and biologically active Actinomycetes screening, phylogenetic analysis.

    Science.gov (United States)

    Li, Z-Y; Liu, Y

    2006-10-01

    The aim of this study was to investigate the bacterial diversity associated with the sponge Craniella australiensis using a molecular strategy and isolating Actinomycetes with antimicrobial potentials. The bacterial diversity associated with South China Sea sponge C. austrialiensis was assessed using a 16S rDNA clone library alongside restriction fragment length polymorphism and phylogenetic analysis. It was found that the C. austrialiensis-associated bacterial community consisted of alpha, beta and gamma-Proteobacteria, Firmicutes, Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully using seawater medium with sponge extracts. According to the BLAST and phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the representative 23 isolates closely matched the Streptomyces sp. while the remaining 12 matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of which 15 are found to possess broad-spectrum antimicrobial potentials. The sponge C. austrialiensis-associated bacterial community is very abundant including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis-associated Actinomycetes have a broad spectrum of antimicrobial activity. This study revealed the diversity of the bacterial community and the isolated Actinomycetes with antimicrobial potentials associated with sponge C. australiensis.

  20. Characterization of Bacterial Community Structure and Diversity in Rhizosphere Soils of Three Plants in Rapidly Changing Salt Marshes Using 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The structure and diversity of the bacterial communities in rhizosphere soils of native Phragmites australis and Scirpus mariqueter and alien Spartina alterniflora in the Yangtze River Estuary were investigated by constructing 16S ribosomal DNA (rDNA) clone libraries. The bacterial diversity was quantified by placing the clones into operational taxonomic unit (OTU) groups at the level of sequence similarity of > 97%. Phylogenetic analysis of the resulting 398 clone sequences indicated a high diversity of bacteria in the rhizosphere soils of these plants. The members of Alphaproteobacteria,Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria of the phylum Proteobacteria were the most abundant in rhizobacteria. Chao 1 nonparametric diversity estimator coupled with the reciprocal of Simpson's index (1/D) was applied to sequence data obtained from each library to evaluate total sequence diversity and quantitatively compare the level of dominance. The results showed that Phragmites, Scirpus, and Spartina rhizosphere soils contained 200, 668, and 382 OTUs, respectively. The bacterial communities in the Spartina and Phragmites rhizosphere soils displayed species dominance revealed by 1/D, whereas the bacterial community in Scirpus rhizosphere soil had uniform distributions of species abundance. Overall, analysis of 16S rDNA clone libraries from the rhizosphere soils indicates that the changes in bacterial composition may occur concomitantly with the shift of species composition in plant communities.

  1. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP.

    Science.gov (United States)

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  2. Identification of dominant bacteria in feces and colonic mucosa from healthy Spanish adults by culturing and by 16S rDNA sequence analysis.

    Science.gov (United States)

    Delgado, Susana; Suárez, Adolfo; Mayo, Baltasar

    2006-04-01

    The aim of this work was to examine by culturing the changes in the total and indicator populations of the feces of two individuals over 1 year and to identify the dominant microbial components of a single sample of feces from each donor. Populations and dominant bacteria from a sample of colonic mucosa from a further individual were also assessed. The culture results were then compared to those obtained with the same samples by 16S rDNA cloning and sequencing. High interindividual variation in representative microbial populations of the gastrointestinal tract (GIT) was revealed by both the culture and the culture-independent techniques. Species belonging to Clostridium clusters (XIVa, IV, and XVIII) predominated in both the fecal and the mucosal samples (except in the mucose cultured isolates), members of Clostridium coccoides cluster XIVa being the most numerous microorganisms. Species of gamma-proteobacteria (Escherichia coli and Shigella spp.), bifidobacteria, and actinobacteria appeared in lower numbers than those of clostridia. From the mucosal cultured sample, only facultative anaerobes and bifidobacteria were recovered, suggesting destruction of the anaerobe population during processing. In accordance with this, the microbial diversity revealed by 16S rDNA sequence analysis was greater than that revealed by culturing. Despite large interindividual differences, distinct human communities may have group-associated GIT microbiota characteristics, such as the low number of Bacteroides seen in the subjects in this study.

  3. Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters

    DEFF Research Database (Denmark)

    Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Larsen, Poul

    A reliable and reproducible method for identification and quantification of the microorganisms involved in biogas production is important for the study and understanding of the microbial communities responsible for the function of anaerobic digester systems. DNA based identification using 16S r...... of the community composition. As such sample specific optimisation and standardisation of DNA extraction, as well PCR primer selection, are essential to minimising the potential for such biases. The aim of this study was to develop a protocol for optimized community profiling of anaerobic digesters. The Fast...

  4. Phylogeny of Deltocephalinae (Hemiptera: Cicadellidae)from China based on partial 16S rDNA and 28S rDNA D2 sequences combined with morphological characters%基于16S rDNA和28S rDNA D2基因序列与形态特征联合分析的中国角顶叶蝉亚科系统发育研究(半翅目:叶蝉科)

    Institute of Scientific and Technical Information of China (English)

    戴仁怀; 陈学新; 李子忠

    2008-01-01

    The phylogeny of 19 genera of Deltocephalinae leafhoppers was analyzed based on 50 adult morphological characters combined with nucleotide sequences of the mitochondrial 16S rDNA and nuclear 28S D2 rDNA genes. One species of Typhlocybinae was included as outgroup. Parsimonian, distance and Bayesian methods were used to estimate the phylogenetic relationships. The topology of the phylogenetic trees generated with different methods was quite similar. We partially resolved the morphologically-defined tribes and the relationships among 19 genera of Deltocephalinae. The genus Macrosteles was well supported to occupy a basal position in the study, so the most primary tribe in Deltocephalinae might be Macrostelini. The phylogenetic analysis trees put all genera of Deltocephalini but Nakaharanus onto a single lineage. The genus Balclutha, corresponding to the tribe Balclnthini,remains unsolved in our analyses. The Euscelini might be a polyphyletic group in the analysis. Analytical result recovered Athysanini and Paralimnini as monophyletic clades. The clade Phlogotettix and Scaphoideus-Nakaharanus was constantly resolved using different methods. We suggested that Scaphoideus, Nakaharanus and Phlogotettix should be included in or into Scaphoideini. But the results resolved poorly the taxonomic status of Xestoeephalini overall.%首次在国内利用28s rDNA D2区段和16s rDNA基因序列,结合50个形态特征对角顶叶蝉亚科(Deltocephalinae)[半翅目(Hemiptera):叶蝉科(cicadellidae)]19个属进行系统发育分析研究.从无水乙醇浸泡保存的标本中提取基因组DNA并扩增了19个内群和1种外群Tyhlocybinae[半翅目(Hemiptera):叶蝉科(cicadelIidae)]种类的28s rDNA D2基因片段并测序,同时扩增了16s rDNA基因片段并测序11条,采用了GenBank中1个种类的16S rDNA同源序列.采用PAuP*4.O和MrBayes3.0两个分析软件和3种建树方法,利用同源28s D2 rDNA和16srDNA两个基因序列与形态特征结合进行系统发

  5. 16s rDNA文库法分析番石榴果实蝇共生菌组成%Composition of symbiotic bacteria associated with Bactrocera correcta analyzed by 16s rDNA libraries

    Institute of Scientific and Technical Information of China (English)

    戴阳; 李志红; 柳丽君; 吴佳教; 邓裕亮

    2012-01-01

    [Objective] The composition of symbiotic bacteria associated with Guava fruit fly, Bacirocera correcta (Bezzi), was investigated to provide the basis for further study on physiological function of the symbionts and co-evolution between bacteria and the host. [Method] 16s rDNA libraries of laboratory population for B. correcta were constructed, and BLAST analysis of bacterial composition and abundance was carried out in this study. [Result] The dominant symbionts in B. correcta male adults were Pseudomonas aeruginosa and Lactococcus lactis, while symbiotic bacteria in female adults were Enterobacter bacteria. [Conclusion] Various bacteria are present in the Guava fruit fly, B. correcta.%[目的]探索分析番石榴果实蝇[ Bactrocera correcta (Bezzi)]的共生菌组成,为进一步探索共生菌的生理功能以及与宿主的协同进化关系奠定基础.[方法]构建番石榴果实蝇室内种群共生菌的16s rDNA文库,BLAST分析其共生菌的组成以及丰度.[结果]室内雄虫共生菌主要为Pseudomonas aeruginosa和Lactococcus lactis,雌虫共生菌主要为Enterobacter属的细菌.[结论]本研究结果证明番石榴果实蝇存在多种共生菌.

  6. Pyrosequencing 检测口腔与胃中的幽门螺杆菌16S rDNA V1区基因序列%Identification of gastric and oral Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 regions

    Institute of Scientific and Technical Information of China (English)

    侯海玲; 孟焕新; 胡伏莲; 成虹

    2005-01-01

    目的通过Pyrosequencing检测口腔与胃幽门螺杆菌16S rDNA V1区序列基因片段分析,以进一步验证口-口传播的的假设.方法选择18例患有慢性中度牙周炎且胃镜活检Hp感染阳性患者及其中10例患者的家属.提取患者胃、牙菌斑和含漱液共74例样本中的DNA,PCR扩增阳性后pyrosequecing 检测16S rDNA V1区序列基因片段.结果患者胃和口腔Hp及其家属口腔Hp的序列相比较,有0~1个碱基不同.结论口腔中的Hp与胃内Hp16S rDNA V1区基因型比较95.8%~100%的同源性,口腔可能为Hp的聚集地.口腔Hp可能与胃Hp感染的复发或再感染有关.

  7. Phylogenetic systematics of Barn Owl (Tyto alba (Scopoli, 1769)) complex inferred from mitochondrial rDNA (16S rRNA) taxonomic implication

    OpenAIRE

    2012-01-01

    The Barn owl, Tyto alba (Scopoli, 1769), occurs worldwide and shows a considerable amount of morphological and geographical variations, leading to the recognition of many subspecies throughout the world. Yet, no comprehensive study has not been done on this species. Data from mitochondrial gene (16S Ribosomal RNA (16S)) with 569 bp length were analyzed for 41 individuals around the world. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis showed two distinct clades includin...

  8. 哈维弧菌16S rRNA基因拷贝数的种内变异%Intra-species variation of 16S rRNA gene copy number of Vibrio harveyi

    Institute of Scientific and Technical Information of China (English)

    郑嘉来; 阎永伟; 唐姝; 杨坤杰; 李长红; 王凯; 张德民

    2015-01-01

    利用荧光定量PCR法测定哈维弧菌( Vibrio harveyi)基因组内16S rRNA基因拷贝数. 基于16S rDNA、pyrH、rpoA和recA4个基因的系统发育学分析鉴定弧菌菌株,然后通过重叠PCR构建包含哈维弧菌模式菌株16S rD-NA和gyrB基因片段的标准质粒,建立了定量PCR测定细胞内16S rRNA基因拷贝数方法,并测定了6株测试菌株和哈维弧菌模式株的16S rRNA基因拷贝数,同时以已知拷贝数的菌株ATCC BAA-1116做对照. 6个测试菌株均为哈维弧菌. 菌株ATCC BAA-1116的16S rRNA基因拷贝数为11个,与已知值相符. 模式菌株MCCC 1H00031T和6个测试菌株SXB-C、SCB-4、NBV00029、NBV00035、NBV00039和NBV00269的拷贝数分别为9、12、13、12、11、13和9个. 利用荧光定量PCR法测定哈维弧菌基因组内16S rRNA基因拷贝数的方法简单可行. 哈维弧菌16S rRNA基因拷贝数的种内变异可能是其适应近岸多变生境的原因之一.%This study aims to determine intra-genomic 16S rRNA gene copy number of Vibrio harveyi strains by a method of fluorescent quantitative PCR. Vibrio strains were identified by multilocussequence analysis based on 16S rDNA, pyrH, rpoA and recA. A standard plasmid, harboring fragments of 16S rDNA and the single copy gene gyrB from V. harveyi type strain, was constructed. A fluorescent quantitative method was established and used to determine the intra-genomic 16S rRNA gene copy number of the tested 6 strains, taken the type strains of V. harveyi and V. harvei ATCC BAA-1116 as reference, whose 16S rRNA gene copy number is well known. All the 6 tested Vibrio strains were identified as V. harveyi. The copy number of 16S rRNA gene in strain ATCC BAA-1116 was 11, consistent with the known values. The copy number of strain MCCC 1H00031T, SXB-C, SCB-4, NBV00029, NBV00035, NBV00039 and NBV00269 was 9, 12, 13, 12, 11, 13 and 9, respectively. Fluorescent quantitative PCR method is simple and reliable for determi-ning 16S rRNA gene copy number

  9. 'Candidatus Phytoplasmas pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

    Science.gov (United States)

    X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rDNA RFLP group 16SrIII, subgroup A. Phylogenetic a...

  10. Accuracy of Conventional PCR Targeting the 16S rRNA Gene with the Ot-16sRF1 and Ot-16sRR1 Primers for Diagnosis of Scrub Typhus: a Case-Control Study.

    Science.gov (United States)

    Kim, Choon-Mee; Cho, Min Keun; Kim, Dong-Min; Yun, Na-Ra; Kim, Seok Won; Jang, Sook Jin; Ahn, Young-Joon; Lim, Donghoon

    2016-01-01

    We retrospectively evaluated the accuracy of conventional PCR targeting the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers for diagnosing scrub typhus. The diagnosis of Orientia tsutsugamushi infection by 16S C-PCR presented an increased sensitivity of 87.0% and specificity of 100% compared with those obtained with other targets and is thus a simple and clinically useful method with good diagnostic accuracy.

  11. Dynamic changes of intestinal 16S rDNA metagenome in 5 infants%动态监测婴儿肠道16S rDNA宏基因组5例报告

    Institute of Scientific and Technical Information of China (English)

    马丽亚; 张敏; 王辉林; 陈睿; 黄艳; 梁小琴; 卢光进

    2014-01-01

    目的:探讨婴儿肠道16S rDNA宏基因组的动态变化。方法收集5例健康婴儿生后3 d、1个月、6个月、1岁时粪便样本共17份,提取细菌总DNA,采用新一代高通量测序技术对16S rDNA的V6高变区测序并进行物种分类、丰度及多样性分析。结果17份样品共产生原始测序数据为2190.66 Mbp,Operational Taxonomic Units(OTU)数量36~308;优势菌门包括变形菌门、厚壁菌门、拟杆菌门和放线菌门;不同婴儿间肠道菌群分布有差异;科水平>1%的物种生后3 d时2~4种,1个月、6个月时增至最多7种,1岁时达10种;4个时间点的npShannon和Simpson指数均值分别为1.117、1.460、2.088、2.50和0.443、0.408、0.229、0.143。结论婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;细菌多样性随日龄而增加,婴儿肠道菌群结构出生后逐渐趋向复杂和多样。%Objective To investigate the dynamic changes of intestinal 16S rDNA metagenome in healthy infants. Methods Seventeen fecal samples were collected at ages of 3 days, 1 month, 6 months and 1 year in 5 infants. Total bacterial DNAs were extracted and submitted high throughout sequencing on the V6 viable region of 16S rDNA. Tags and Operational Taxonomic Units (OTU) were then obtained and analysis of taxonomy, abundance and alpha diversity were performed. Results In total 2 190.66 Mbp raw data in 17 samples were produced. The OTU numbers ranged from 36 to 308. The dominate phylum included Proteobacteria, Firmicutes and Bacteroidetes and Actinobacteria. The bacterial families>1%increased from only 2-4 per sample on day 3 to 7 at 1 or 6 months, 10 at 12 months. The average npShannon and Simpson index on day 3, at 1 month, 6 months and 1 year were 1.117, 1.460, 2.088, 2.50 and 0.443, 0.408, 0.229, 0.143 respectively. Conclusions Infants’ intestines harbor abounding bacterial genomes. Distinct individual differences exist in infants in terms of

  12. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods

    DEFF Research Database (Denmark)

    On, Stephen L.W.; Harrington, C.S.

    2001-01-01

    Aims: To assess the efficacy of numerical analysis of PFGE-DNA profiles for identification and differentiation of Campylobacter fetus subspecies. Methods and Results: 31 Camp. fetus strains were examined by phenotypic, PCR- and PFGE-based methods, and the 16S rDNA sequences of 18 strains compared....... Numerical analysis of PFGE-DNA profiles divided strains into two clusters at the 86% similarity level. One cluster contained 19 strains clearly identified as Camp. fetus subsp. venerealis. The other cluster comprised 12 strains, of which 10 were unambiguously identified as Camp. fetus subsp. fetus....... The remaining two strains were identified as Camp. fetus subsp. venerealis by either phenotypic or PCR methods, but not both. At higher similarity levels, clusters containing isolates from each of two countries were identified, suggesting that certain clones predominate in certain geographical regions...

  13. 应用16S rDNA全序列同源性分析鉴定致羔羊脑炎性粪肠球菌%Rapid identification of Enterococcus faecalis causing lambs' encephalitis by homology analysis of 16S rDNA complete sequence

    Institute of Scientific and Technical Information of China (English)

    齐亚银; 王静梅; 张莉; 剡根强

    2011-01-01

    选用通过常规生理生化特性鉴定的疑似粪肠球菌菌株3株,采用16S rDNA的通用引物对其可变区基因进行克隆、测序和同源性比对,并用CLUSTAL和MEGA软件分析其遗传距离并构建系统进化树.结果表明,此3株菌株通过同源性比对,与GenBank数据库中粪肠球菌的同源性均大于99.8%,经遗传距离分析和系统进化树的构建均表明为粪肠球菌.肠球菌属细菌有40个种,各种之间的生化生理特性差别甚微,在无法通过生化生理特性准确鉴定到种的情况下,用粪肠球菌的16S rDNA全序列的测定及系统进化树的分析是鉴定该菌的一个科学可靠的方法.%To identify rapidly Enlerococcus faecalis causing lambs' encephalitis by 16S rDNA complete sequence ho-mology analysis, 3 isolates doubting Enterococcus faecalis were selected by conventional physiological and biochemical characterization. A variable region gene was amplified by 16S rDNA primers, then cloned, sequenced and analyzed homologous of sequences. Using CLUSTAL and MEGA software, we analyzed pair-wise divergence and constructed the phylogenetic trees. Because the results showed that the homology of 3 isolates with Enterococcus faecalis were 99. 8% ,100% and 100%(>97%) respectively. By genetic distance analysis and phylogenetic tree construction, the three isolates were all Enterococcus faecalis. Because the 40 genuses of Enterococcus have similar physiological and biochemical characteristics,using 16S rDNA complete genomic sequencing and phylogenetic tree analysis for identification of Enterococcus faecalis is a" feasible method.

  14. [16S rDNA diversity analysis of 30 Streptomycetes isolates displaying significant cytotoxic activity against B16 cell from near-shore sediments of Hainan Island].

    Science.gov (United States)

    Yan, Li-Ping; Hong, Kui; Hu, Shen-cai; Liu, Li-hua

    2005-04-01

    A total of 354 isolates of actinomycetes, of which 76 were detected cytotoxic activity was isolated from near-shore marine samples collected at Wenchang mangrove, DanZhou harbor and YanPu harbor. Four isolation methods were employed, which are SDS pretreatment, phenol pretreatment, heating pretreatment and potassium dichromate selection culture, and media such as'Yeast extract-Malt extract (YE), Glucose-Asprine (GA), Starch-Casin (SC), Starch-KNO3 (Gause) were used. It was showed that heating pretreatment and potassium dichromate selection culture were more considerable methods for extensive isolation of actinomycetes. Medium YE and Gause showed best results in both the total number of actinomycetes and the number of active isolates against tumor cell B16. The genotypic diversity of 30 strains of Streptomycetes possessing strong cytotoxic activity against B16 cell (ID50 > or =200) was analyzed by 16S ARDRA, which resulted in 17 RFLP types, and indicated relatively rich genotypic diversity among these Streptomycetes. 16S rDNA sequence analysis of three strains, 050642, 060386 and 060524 (ID50 > or = 1200) further confirmed that they all belong to Streptomyces genus and strain 050642 was suggested a novel Streptomyces. Spp with the highest similarity of 95% to Streptomyces cattleya.

  15. Studies on biodegradation and molecular characterization of 2,4-D Ethyl Ester and Pencycuron induced Cyanobacteria by using GC-MS and 16S rDNA sequencing

    Directory of Open Access Journals (Sweden)

    J. I. Nirmal Kumar

    2013-03-01

    Full Text Available GC-MS study and molecular characterization by 16S rDNA amplification were carried out to evaluate differential effects of 2,4-D ethyl ester and pencycuron on Anabaena fertilissima, Aulosira fertilissima and Westiellopsis prolifica. Each organism has its own capacity to degrade both pesticides into various subgroups depending largely upon the main functional group of each individual pesticide. Hence, different subgroups like 2,4-D methyl ester, 2,4-D isobutyl ester, Isobutyric acid allyl ester, 3-Bromobutyric acid, 2,4-D butyl ester, Hydroxyurea, Trifluroacetic acid, 2-Methyl propyl ester, Acetic acid 2-propenyl ester and Acetic acid (2,3-dichlorophenoxy were transformed from 2,4-D ethyl ester while Benzoxazole was the only compound generated from pencycuron treated W. prolifica. The results obtained by 16S rDNA sequencing confirmed that 16S rDNA region of Anabaena fertilissima was more affected by 2,4-D ethyl ester as there was no homology in the region of 39 basepairs, in addition, several mismatches and gaps were observed, whereas less difference in 16S rDNA was observed in case of Aulosira fertilissima and W. prolific on forth day. However, there was no significant change in the sequence of 16S rDNA pattern of all the three test organisms after 16-days of exposure to pencycuron treatment.

  16. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, S. de; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  17. Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, de S.; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  18. Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing.

    Science.gov (United States)

    Li, Xin; Zhu, Xiao-fei; Zhang, Cheng-fei; Cathro, Peter; Seneviratne, C J; Shen, Song

    2013-02-01

    Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions, with no information available for the Chinese population. As such, we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing, China using 16S rDNA gene sequencing techniques. Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction. Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected. Purified plasmid DNA with 16S rDNA gene inserts was sequenced, and the sequences were searched against GenBank databases using the BLASTN algorithm. Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping. More than 150 taxa were obtained. Primary endodontic infection was mainly associated with Burkholderia cepacia, Actinomyces, Aranicola spp. and Streptococcus sanguinis, whilst Burkholderia cepacia was predominant in the persistent endodontic infections. There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beijing in comparison to other geographical or ethnic reports.

  19. Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

    Institute of Scientific and Technical Information of China (English)

    LI Xin; ZHU Xiao-fei; ZHANG Cheng-fei; Peter Cathro; CJ Seneviratne; SHEN Song

    2013-01-01

    Background Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions,with no information available for the Chinese population.As such,we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing,China using 16S rDNA gene sequencing techniques.Methods Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction.Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected.Purified plasmid DNA with 16S rDNA gene inserts was sequenced,and the sequences were searched against GenBank databases using the BLASTN algorithm.Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping.Results More than 150 taxa were obtained.Primary endodontic infection was mainly associated with Burkholderia cepacia,Actinomyces,Aranicola spp.and Streptococcus sanguinis,whilst Burkholderia cepacia was predominant in the persistent endodontic infections.Conclusion There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beiiing in comoarison to other geographical or ethnic reports.

  20. 16S rDNA在乳酸菌菌种鉴定及聚类分析上的应用%Application of 16S rDNA fingerprint on the strain identification and traceability isolated from yogurts

    Institute of Scientific and Technical Information of China (English)

    杨柳; 杨捷琳; 谌鸿超; 曹敏仪; 潘良文

    2013-01-01

    Microecological products are paid more and more attention.Most commercial microecological products on local market are the yogurts which contain lactic acid bacteria.The common technology of strain identification are the physiological and biochemical test or the application of PCR with some defects such as time-consuming or need specialized person.We used Riboprinter to complete the subspecies level of identification of 16 lactobacillus bacterias isolated from 11 yogurt samples based on the separation and purification of the bacterias.Not only chose the optimization of the culture conditions,but also established the "Traceability File" used for the quality research.What's more,the use of the 16s rDNA fingerprint analysis and real time PCR helped to confirm the verification of the results from the colony identification.%近年来,微生态制剂日益受到市场关注.市售微生态食品以益生菌酸奶或乳酸饮料为主,其中活的乳酸菌含量及菌株特性是决定微生态制剂质量的关键.目前常用的菌种鉴定技术包括生理生化方法或PCR方法,前者工作量大周期长,后者需要专业人员操作,且难以区分死活细菌.本文选取了11种市售的乳酸菌产品,采用MC、MRL、TPY等乳酸菌专用培养基进行乳酸菌计数及菌种分离,进一步用荧光定量PCR方法验证了产品中常规培养法难以分离的双歧杆菌,在此基础上,采用Riboprinter基因指纹图谱分析仪,对分离自11种酸奶样品的16株乳酸菌菌进行了核酸水平的鉴定,建立了16S rDNA聚类分析分子图谱,方便并简化对微生态产品的质量进行“物证相符”的考证,初步分析了目前市场上市售酸奶所用菌种的种属相关性.

  1. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene.

    Science.gov (United States)

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-07-27

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese's complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes.

  2. COMPARACIÓN ENTRE EL POTENCIAL DE LAS REGIONES VARIABLES DEL 16S rDNA PARA LA IDENTIFICACIÓN DE Lactobacillus spp. (LACTOBACILLIACEAE

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    LAURA N. GONZÁLEZ GARCÍA

    2013-01-01

    Full Text Available El 16s rDNA es utilizado para la identificación bacteriana dada su tasa de variación entre especies. Algunas de las regiones variables de la subunidad ribosomal son más informativas que otras por lo cual en este estudio se evalúa el potencial de identificación aportado por cada región y combinaciones entre ellas. Se extrajeron las regiones variables V1 a la V8 del 16s rDNA de diferentes cepas y especies de Lactobacillus y se analizaron mediante los paquetes de STAP (ss-RNA Taxonomy Assigning Pipeline y RDP (Ribosomal Database Project multiclassifier. Adicionalmente se evaluaron árboles filogenéticos de máxima verosimilitud. Nuestros resultados muestran que la mayoría de regiones variables logran dar una correcta clasificación hasta género, sin embargo no son suficientes para clasificar hasta especie usando STAP. La región que presenta el mayor número de amplímeros es V5V6, sin embargo es la que presenta la mayor cantidad de falsos negativos. La que presenta el mayor número de verdaderos positivos es V1V3 (especie para STAP y V5V8(género para RDP. Las filogenias evaluadas mostraron que la topología de referencia se puede obtener con diferentes combinaciones de regiones variables e.g., V1V3 y V1V8. El estudio experimental de las cepas contenidas en un tampón comercial mostró que el amplicón V1V8 y el V1V3 dan una misma clasificación correcta. Proponemos la región V1V3 como la región mínima para clasificación correcta de Lactobacillus spp.. En conclusión, la región mínima para clasificar especies del género Lactobacillus es la V1V3, la cual es útil para estudios meta- genómicos de muestras de probióticos.

  3. Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: a case control study

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    Nakstad Britt

    2009-01-01

    Full Text Available Abstract Background Early onset bacterial sepsis is a feared complication of the newborn. A large proportion of infants admitted to the Neonatal Intensive Care Unit (NICU for suspected sepsis receive treatment with potent systemic antibiotics while a diagnostic workup is in progress. The gold standard for detecting bacterial sepsis is blood culture. However, as pathogens in blood cultures are only detected in approximately 25% of patients, the sensitivity of blood culture is suspected to be low. Therefore, the diagnosis of sepsis is often based on the development of clinical signs, in combination with laboratory tests such as a rise in C – reactive protein (CRP. Molecular assays for the detection of bacterial DNA in the blood represent possible new diagnostic tools for early identification of a bacterial cause. Methods A broad range 16S rDNA polymerase chain reaction (PCR without preincubation was compared to conventional diagnostic work up for clinical sepsis, including BACTEC blood culture, for early determination of bacterial sepsis in the newborn. In addition, the relationship between known risk factors, clinical signs, and laboratory parameters considered in clinical sepsis in the newborn were explored. Results Forty-eight infants with suspected sepsis were included in this study. Thirty-one patients were diagnosed with sepsis, only 6 of these had a positive blood culture. 16S rDNA PCR analysis of blinded blood samples from the 48 infants revealed 10 samples positive for the presence of bacterial DNA. PCR failed to be positive in 2 samples from blood culture positive infants, and was positive in 1 sample where a diagnosis of a non-septic condition was established. Compared to blood culture the diagnosis of bacterial proven sepsis by PCR revealed a 66.7% sensitivity, 87.5% specificity, 95.4% positive and 75% negative predictive value. PCR combined with blood culture revealed bacteria in 35.1% of the patients diagnosed with sepsis

  4. Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

    Science.gov (United States)

    Wood, Jacqueline; Scott, Karen P.; Avguštin, Gorazd; Newbold, C. James; Flint, Harry J.

    1998-01-01

    We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples. PMID:9758785

  5. Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

    Science.gov (United States)

    Lee, I M; Bartoszyk, I M; Gundersen-Rindal, D E; Davis, R E

    1997-01-01

    A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera. PMID:9212413

  6. Study on bacterial population diversity in raw milk with 16S rDNA library analysis method%16S rDNA克隆文库法分析生鲜牛乳中细菌种群的多样性

    Institute of Scientific and Technical Information of China (English)

    张敏爱; 张建军; 王子亮; 苑利; 杨秀清

    2014-01-01

    目的:利用16S rDNA基因文库分析法,对生鲜牛乳中微生物的种群多样性进行研究。方法提取生鲜牛乳中总微生物基因组DNA为模板,将扩增到的生鲜牛乳样品的16S rDNA的PCR产物与pMD~18T载体连接,构建其菌群的16S rDNA文库。对随机选取的56个阳性克隆子进行序列测定和BLAST比对。结果文库序列分析表明,有53个克隆子分属3个不同的类群,即厚壁菌类群、变形菌类群和异常球菌-栖热菌类群,其余3个克隆子属于未知类群。其中,优势细菌类群为厚壁菌类群(86%),在该类群中,尤以无乳链球菌为主要代表,约占所有克隆子的82%;其他类群为γ~变形菌类群(7.1%)和异常球菌-栖热菌门类群(1.8%)。系统发育分析表明,未知类群在分类地位上更接近与厚壁菌类群。结论生鲜牛乳中微生物具有多样性,无乳链球菌为其中的优势种群,同时生鲜牛乳中还存在葡萄球菌等致病菌,或不动杆菌和肠道菌等条件致病菌。%Objective Using 16S rDNA gene clone library analysis method, the bacterial population di-versity in raw milk was analyzed.Methods Microbial genomic DNA in raw milk was extracted and used as template to analysis.PCR products of 16S rDNA in raw milk were ligated with vector pMD~18T, constructing a bacterial gene clone library. 56 positive clones were picked randomly from the 16S rDNA library, followed by sequence analysis and BLAST comparison.Results Sequence analysis showed that 53 clones could be di-vided into 3 groups:Firmicutes ,Proteobacteria , andDeinococcus~Thermus. The rest of 3 clones were uncul-tured bacteria. The dominant group belonged toFirmicutes(86%), in whichStreptococcusagalactiae predo-minated, and possessed 82% of all clone in the library. Other groups wereγ~Proteobacteria (7.1%) andDei- nococcus~Thermus (1.8%). And the uncultured bacteria were closed toFirmicutes in the phylogenetic tree. Conclusion The bacterial

  7. Lactic acid bacterial diversity in the traditional mexican fermented dough pozol as determined by 16S rDNA sequence analysis.

    Science.gov (United States)

    Escalante, A; Wacher, C; Farrés, A

    2001-02-28

    The lactic acid bacteria diversity of pozol, a Mexican fermented maize dough, was studied using a total DNA extraction and purification procedure and PCR amplification of 16S rDNA for gram-positive and related bacterial groups. Thirty-six clones were obtained and sequenced to 650 nucleotides. These partial sequences were identified by submission to the non-redundant nucleotide database of NCBI. The identified sequences were aligned with reference sequences of the closest related organisms. This analysis indicated that only 14 sequences were unique clones and these were identified as Lactococcus lactis, Streptococcus suis, Lactobacillus plantarum, Lact. casei, Lact. alimentarium, and Lact. delbruekii and Clostridium sp. Two non-ribosomal sequences were also detected. Unlike other environments analyzed with this molecular approach where many unidentified microorganisms are found, the identity of most sequences could be established as lactic acid bacteria, indicating that this is the main group among the gram-positive bacteria in pozol. Use of this molecular method permitted detection of lactic acid bacteria different from those previously isolated and identified by culture techniques

  8. Community analysis of ammonia oxidizer in the oxygen-limited nitritation stage of OLAND system by DGGE of PCR amplified 16S rDNA Fragments and FISH

    Institute of Scientific and Technical Information of China (English)

    ZHANG Dan; ZHANG De-min; LIU Yao-ping; CAO Wen-wei; CHEN Guan-xiong

    2004-01-01

    OLAND(oxygen limited autotrophic nitrification and denitrification) nitrogen removal system was constructed by coupling with oxygen limited nitritation stage and anaerobic ammonium oxidation stage. Ammonia oxidizer, as a kind of key bacteria in N cycle, plays an important role at the oxygen limited nitritation stage of OLAND nitrogen removal system. In this study, specific amplification of 16S rDNA fragment of ammonia oxidizer by nested PCR, separation of mixed PCR samples by denaturing gradient gel electrophoresis(DGGE), and the quantification of ammonia oxidizer by Fluorescence in situ hybridization(FISH) were combined to investigate the shifts of community composition and quantity of ammonia oxidizer of the oxygen limited nitritation stage in OLAND system. It showed that the community composition of ammonia oxidizer changed drastically when dissolved oxygen was decreased gradually, and the dominant ammonia oxidizer of the steady nitrite accumulation stage were completely different from that of the early stage of oxygen limited nitritation identified by DGGE . It was concluded that the Nitrosomonas may be the dominant genus of ammonia oxidizer at the oxygen limited nitritation stage of OLAND system characterized by nested PCR-DGGE and FISH, and the percentage of Nitrosomonas was 72.5% ( 0.8% of ammonia oxidizer at the steady nitrite accumulation stage detected by FISH.

  9. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

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    Byeng R. Min

    2014-01-01

    Full Text Available Eighteen Kiko-cross meat goats (n=6 were used to collect gastrointestinal (GI bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB. Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS; 0.17% condensed tannins (CT dry matter (DM; 15% PB and 15% WS (1.6% CT DM, and 30% PB and 0% WS (3.2% CT DM. A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB to 46.5% (control and 47.1% (15% PB. Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%, Methanosphaera (3.3, 2.3, and 3.4%, and Methanobacteriaceae (1.2, 0.6, and 0.7% population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P=0.05 with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  10. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing.

    Science.gov (United States)

    Min, Byeng R; Solaiman, Sandra; Shange, Raymon; Eun, Jong-Su

    2014-01-01

    Eighteen Kiko-cross meat goats (n = 6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucidate changes in GI bacteria and methanogenic archaea diversity among the diets. Proteobacteria was the most dominant phylum in goats with mean relative abundance values ranging from 39.7 (30% PB) to 46.5% (control) and 47.1% (15% PB). Other phyla individually accounted for fewer than 25% of the relative abundance observed. Predominant methanogens were Methanobrevibacter (75, 72, and 49%), Methanosphaera (3.3, 2.3, and 3.4%), and Methanobacteriaceae (1.2, 0.6, and 0.7%) population in control, 15, and 30% PB, respectively. Among methanogens, Methanobrevibacter was linearly decreased (P = 0.05) with increasing PB supplementation. These results indicate that feeding PB selectively altered bacteria and methanogenic archaeal populations in the GI tract of goats.

  11. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP

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    Sun Yan

    2008-07-01

    Full Text Available Abstract Background The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows. Results Ubiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica and 6 cows were positive for Campylobacter spp. (tentative lanienae. Conclusion Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.

  12. Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents.

    Science.gov (United States)

    Kodjo, A; Villard, L; Veillet, F; Escande, F; Borges, E; Maurin, F; Bonnod, J; Richard, Y

    1999-02-01

    Pasteurella pneumotropica is an opportunistic bacterium frequently isolated from colonies of various laboratory rodents. Identification of this species, including its differentiation into two distinct biotypes (Jawetz and Heyl), is usually based on the use of conventional bacteriologic methods. In this study, a 16S rDNA fragment amplification procedure was developed for use as an alternative method for identification and differentiation of P. pneumotropica. Polymerase chain reaction (PCR) products were two distinctive fragments of 937 and 564 bp specific for biotypes Jawetz and Heyl, respectively. Specificity of PCR products could be achieved by EcoRI cleavage, leading to 596 plus 341-bp and 346 plus 218-bp fragments for each of the amplification products. Use of this procedure confirmed identification of 34 field isolates and allowed definitive identification of some strains that could not have been done by use of bacteriologic examinations. Field isolates subjected to random amplified polymorphic DNA (RAPD) analysis had high genetic diversity among biotype Jawetz strains in contrast to biotype Heyl strains. In conclusion, RAPD could represent an additional means for identification of ambiguous strains of biotype Heyl and a valuable epidemiologic tool for identification of biotype Jawetz strains of P. pneumotropica.

  13. Atmospheric Deposition-Carried Zn and Cd from a Zinc Smelter and Their Effects on Soil Microflora as Revealed by 16S rDNA

    Science.gov (United States)

    Shen, Feng; Li, Yanxia; Zhang, Min; Awasthi, Mukesh Kumar; Ali, Amjad; Li, Ronghua; Wang, Quan; Zhang, Zengqiang

    2016-12-01

    In this study, we investigated the influence of heavy metals (HM) on total soil bacterial population and its diversity pattern from 10 km distance of a Zinc smelter in Feng County, Qinling Mountain, China. We characterized and identified the bacterial community in a HM polluted soil using 16S rDNA technology. Out results indicated that the maximum soil HM concentration and the minimum bacterial population were observed in S2 soil, whereas bacterial diversity raised with the sampling distance increased. The bacterial communities were dominated by the phyla Proteobacteria, Acidobacteria and Actinobacteria in cornfield soils, except Fimicutes phylum which dominated in hilly area soil. The soil CEC, humic acid (HA)/fulvic acid (FA) and microbial OTUs increased with the sampling distance increased. Shewanella, Halomonas and Escherichia genera were highly tolerant to HM stress in both cultivated and non-cultivated soil. Finally, we found a consistent correlation of bacterial diversity with total HM and SOM along the sampling distance surrounding the zinc smelter, which could provide a new insight into the bacterial community-assisted and phytoremediation of HM contaminated soils.

  14. Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

    Science.gov (United States)

    Kembel, Steven W.; Wu, Martin; Eisen, Jonathan A.; Green, Jessica L.

    2012-01-01

    The abundance of different SSU rRNA (“16S”) gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly – from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data. PMID:23133348

  15. Phylogenetic systematics of Barn Owl (Tyto alba (Scopoli, 1769 complex inferred from mitochondrial rDNA (16S rRNA taxonomic implication

    Directory of Open Access Journals (Sweden)

    Mansour Aliabadian

    2012-09-01

    Full Text Available The Barn owl, Tyto alba (Scopoli, 1769, occurs worldwide and shows a considerable amount of morphological and geographical variations, leading to the recognition of many subspecies throughout the world. Yet, no comprehensive study has not been done on this species. Data from mitochondrial gene (16S Ribosomal RNA (16S with 569 bp length were analyzed for 41 individuals around the world. Maximum likelihood (ML, maximum parsimony (MP and Bayesian analysis showed two distinct clades including alba clad (old world and furcata clad (new world. The amount of genetic variation within each of these clades ranged from 0.5-1.7 but variation between clades was 3.7. This data may suggest that Barn owls of the Old World may be a separate species from those of the New World.

  16. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    Science.gov (United States)

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis.

  17. 一株抗铜根瘤菌的分离鉴定及其16S rDNA序列分析%Isolation,Identification and 16S rDNA Sequences Analysis of a Rhizobium Strain Resistant to Copper

    Institute of Scientific and Technical Information of China (English)

    樊连梅; 李哲斐; 韦革宏

    2011-01-01

    为了发挥微生物和植物在重金属矿尾区铜污染土壤的共同修复作用,本研究从陕西省凤县矿尾区天蓝苜蓿根瘤中筛选分离到一株抗重金属铜的菌株,定名为CCNWSX0020.该菌株在YMA琼脂平板上抗铜浓度为3.0mmol/L,TY液体培养基中抗铜浓度为1.4 mmo1/L,因此,它是研究根瘤菌抗铜机制的良好菌株.该菌株经生理生化分析、G+C mo1%测定、DNA同源性分析及16S rDNA序列相似性比较,鉴定为苜蓿中华根瘤菌(Sinorhizobium meliloti),它与苜蓿中华根瘤菌LMG6133的菌株(GenBank登录号为X67222)相似性为99.7%.由于根瘤菌具有独特的生物学地位,即能够与豆科植物形成共生体,这为进一步构建耐重金属的生物体系提供了良好的遗传基础,具有潜在的生态效益和社会效益.%In recent years ,with the development of mining, smelting and processing of copper industry, soil copper pollution is becoming increasingly serious. Thus it is imperative to restore the soil copper contamination. In this study, in order to develop collaborative restoration activity of microorganisms and plants in the copper-contaminated soils of heavy metals tailing area, an excellent Rhizobium strain resistance to copper was screened from mining tailings Fengxlan county,Shaanxi province. The strain,named CCNWSX0020,resisted to 3.0 mmol/L Cu2+ on YMA agar plate and to 1.4 mmol/L Cu2+ in TY liquid medium, so it is an important strain using to study the resistant copper mechanisms of microbe. It was preliminarily identified as a strain of Sinorhizobium meliloti, based on its physiological and biochemical characteristics analysis, including G + C mol% determination, DNA homology hybridization and 16S rDNA sequence similarity comparison. The similarity between CCNWSX0020 and S. meliloti LMG6133 ( GenBank accession number X67222) was 99.7%. Because of Rhizobia with the unique biology status that is being able to form symbiont with legume,which can provide a

  18. Tolerance to Lead-zinc Stress and 16S rDNA PCR-RFLP of Rhizobia Isolated from Nodules of Leguminous Plants in Huize Lead-zinc Mining Tailings%会泽铅锌尾矿区豆科植物根瘤菌的耐铅锌及16S rDNA PCR-RFLP研究

    Institute of Scientific and Technical Information of China (English)

    缪福俊; 熊智; 李彪; 龚秀会; 孙浩

    2011-01-01

    对21株分离自会泽铅锌尾矿区豆科植物根瘤菌进行耐复合铅锌双盐抗逆性及16S rDNA PCR-RFLP研究,结果表明,该区的豆科植物根瘤菌对铅锌双盐具有良好的耐性能力,筛选出HSY6和HZH2两株抗性能力强的菌株,分别与三叶草、紫花苜蓿共生.21株供试菌株的16S rDNA PCR-RFLP在73%的相似水平上分为5个遗传群,分别为慢生根瘤菌属(2株)、中慢生根瘤菌属(5株)、中华根瘤菌属(1株)、土壤杆菌属(3株)、根瘤菌属(10株).供试根瘤菌类群对重金属铅锌的耐性为慢生根瘤菌属>中慢生根瘤菌属>中华根瘤菌属>土壤杆菌属>根瘤菌属.%The double lead-zinc tolerance of 21 rhizobia strains -were studied for the sake of using Leg-ume-rhizobia symbiosis to improve ecological environment in Huize lead-zinc mining tailings. The results showed that The results showed that the capability to tolerate double lead and zinc was strong. 2 stains of HSY6 isolated from nodules of Trifolium repens ,HZH2 isolated from nodules of Lespedza formosa showed the highest tolenrance to lead-zinc. The dendrogram of 21 rhizobia strains 16S rDNA PCR-RFLP fingerprinting were revealed that there are five genetic groups at 73% similarity level. Group 1 is Bradyhizobium (2 strains). Group 2 is Mesorhizobium (5 strains). Group 3 is Sinorhizobi-um (1 strain). Group 4 is Agrobacterium (3 strains). Group 5 Rhizobium (10 strains). The sequence of genetic groups to tolerate double lead and zinc salt is that:Bradyhizobium>Mesorhizobium>Sino-rhizobium> Agrobacterium> Rhizobium.

  19. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Science.gov (United States)

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  20. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    Directory of Open Access Journals (Sweden)

    Huijing Hao

    Full Text Available API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  1. Phylogenetic relationships between Bacillus species and related genera inferred from 16s rDNA sequences Relações filogenéticas entre espécies de Bacillus e gêneros relacionados baseadas em sequencias 16S rDNA

    Directory of Open Access Journals (Sweden)

    Mi Sun Wei Wang

    2009-09-01

    Full Text Available Neigh-borjoining, maximum-parsimony, minimum-evolution, maximum-likelihood and Bayesian trees constructed based on 16S rDNA sequences of 181 type strains of Bacillus species and related taxa manifested nine phylogenetic groups. The phylogenetic analysis showed that Bacillus was not a monophyletic group. B. subtilis was in Group 1. Group 4, 6 and 8 respectively consisted of thermophiles, halophilic or halotolerant bacilli and alkaliphilic bacilli. Group 2, 4 and 8 consisting of Bacillus species and related genera demonstrated that the current taxonomic system did not agree well with the 16S rDNA evolutionary trees. The position of Caryophanaceae and Planococcaceae in Group 2 suggested that they might be transferred into Bacillaceae, and the heterogeneity of Group 2 implied that some Bacillus species in it might belong to several new genera. Group 9 was mainly comprised of the genera (excluding Bacillus of Bacillaceae, so some Bacillus species in Group 9: B. salarius, B. qingdaonensis and B. thermcloacae might not belong to Bacillus. Four Bacillus species, B. schlegelii, B. tusciae, B. edaphicus and B. mucilaginosus were clearly placed outside the nine groups.Árvores utilizando os métodos de neighbor-joining, máxima parcimônia, evolução mínima, máxima verossimilhança e bayesiana, construídas baseadas em seqüências de rDNA 16S de 181 linhagens-tipo de espécies de Bacillus e taxa relacionados, mostraram a formação de nove grupos filogenéticos. A análise filogenética mostrou que Bacillus não é um grupo monofilético. B. subtilis se colocou no Grupo 1. Grupos 4, 6 e 8, respectivamente, consistiram de bacilos termofílicos, halofílicos ou halotolerantes e alcalifílicos. Grupos 2, 4 e 8 consistindo de espécies de Bacillus e gêneros relacionados demonstraram que o sistema taxonômico corrente não concorda perfeitamente com as árvores evolucionárias por rDNA 16S. A posição de Caryophanaceae e Planococcaceae no Grupo 2 sugere

  2. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    Directory of Open Access Journals (Sweden)

    S.M. Azwai

    2016-03-01

    Full Text Available The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk. Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6% yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS with culture characteristics of Vibrio spp. More than half (n=27 of processed seafood samples (n=46 yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 х104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 х104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9% were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  3. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

    Science.gov (United States)

    Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  4. Preliminary evaluation of the use of soil bacterial 16S rDNA DNA markers in sediment fingerprinting in two small endorheic lagoons in southern Spain

    Science.gov (United States)

    Gomez, Jose Alfonso; Landa del Castillo, Blanca; Guzman, Gema; Petticrew, Ellen L.; Owens, Phillip N.

    2016-04-01

    bulk community of DNA was extracted from 250 mg of soil samples (three replicates per sample) using the procedure described in Landa et al. (2014). The bacterial 16S rRNA gene V1-V2 hypervariable regions were amplified in polymerase chain reaction (PCR). The sequencing procedure was performed according to the manufacturer's recommendations using MiSeq Reagent Kit v2 for 300 cycles on MiSeq desktop sequencer. The raw dataset for each sample consisted of the number of counts for each of the 6640 operational taxonomic units (OTU) analyzed. All the screening and analysis was performed independently for each lagoon. Given the large number of OTUs, a first screening was made discarding any OTU that did not presented at least five samples with counts >20 for that OTU. This lowered the number of OTUs to 205 in Dulce and 217 in Zoñar. Because of the limited number of samples, we did not perform independent analysis for each soil depth. All the analyses were performed twice; one with the original number of counts and another with the normalized number of counts. We screened the OTU following a 4-step method to determine those with the best ability to discriminate among the three potential source areas. These steps were: 1) eliminate OTUs with no readings or very few, that could be experimental noise; 2) keep only OTUs that are different among source areas; 3) eliminate OTUs that range outside of feasible solutions to explain average values found in sediment; and 4) eliminate OTUs with the largest variability. Afterwards, several over-determined mixing models were solved considering different combinations of OTUs using limSolve (Soetaert et al., 2014) in R. Preliminary results show that 0.2 to 0.6 % of the searched OTUs (i.e. 14 to 42) had the potential for use in the mixing models after the four-step screening process. The results indicate a large variability in the number of counts among the samples from different areas within the subcatchments ranging, on average, from 49 to

  5. Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor.

    Science.gov (United States)

    Rintala, Anniina; Pietilä, Sami; Munukka, Eveliina; Eerola, Erkki; Pursiheimo, Juha-Pekka; Laiho, Asta; Pekkala, Satu; Huovinen, Pentti

    2017-04-01

    Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3-V4 and the other targeting the V4-V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results.

  6. Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

    Science.gov (United States)

    Rintala, Anniina; Pietilä, Sami; Munukka, Eveliina; Eerola, Erkki; Pursiheimo, Juha-Pekka; Laiho, Asta; Pekkala, Satu; Huovinen, Pentti

    2017-01-01

    Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3–V4 and the other targeting the V4–V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results. PMID:28260999

  7. Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ying; Sunny Aiyuk; XU Hui; CHEN Guanxiong; Willy Verstraete

    2005-01-01

    The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.

  8. Linkage disequilibrium mapping places the gene causing familial Mediterranean fever close to D16S246

    Energy Technology Data Exchange (ETDEWEB)

    Levy, E. N.; Aksentijevich, I.; Pras, E. [National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD (United States)] [and others

    1996-03-01

    This report presents refined genetic mapping data for the gene causing familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation. We sampled 65 Jewish, Armenian, and Arab families and typed them for eight markers from chromosome 16p. Using a new algorithm that permits multipoint calculations for a dense map of markers in consanguineous families, we obtained a maximal LOD score of 49.2 at a location 1.6 cM centromeric to D16S246. A specific haplotype at D16S283-D16S94-D16S246 was found in 76% of Moroccan and 32% of non-Moroccan Jewish carrier chromosomes, but this haplotype was not overrepresented in Armenian or Arab FMF carriers. Moreover, the 2.5-kb allele at D16S246 was significantly associated with FMF in Moroccan and non-Moroccan Jews but not in Armenians or Arabs. Since the Moroccan Jewish community represents a relatively recently established and genetically isolated founder population, we analyzed the Moroccan linkage-disequilibrium data by using Luria-Delbruck formulas and simulations based on a Poisson branching process. These methods place the FMF susceptibility gene within 0.305 cM of D16S246 (2-LOD-unit range 0.02-0.64 cM). 41 refs., 3 figs., 5 tabs.

  9. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

    Directory of Open Access Journals (Sweden)

    Chiari Ylenia

    2005-03-01

    Full Text Available Abstract Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%, with low degrees of pairwise haplotype divergence within populations (0–1%. Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

  10. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

  11. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Science.gov (United States)

    Wang, Yong; Qian, Pei-Yuan

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  12. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Bacterial 16S ribosomal DNA (rDNA amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90% were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  13. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers

    NARCIS (Netherlands)

    Zwart, G.; Crump, B.C.; Kamst-van Agterveld, M.P.; Hagen, F.; Han, S.K.

    2002-01-01

    In order to identify patterns in bacterial community composition in freshwater habitats, we analyzed the available database of 16S rDNA sequences from freshwater plankton, including 24 new sequences from Parker River (Massachusetts, USA), 42 from Lake Soyang (South Korea) and 148 from Lake IJssel

  14. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    Science.gov (United States)

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  15. Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children.

    Science.gov (United States)

    Rosey, Anne-Laure; Abachin, Eric; Quesnes, Gilles; Cadilhac, Céline; Pejin, Zagorka; Glorion, Christophe; Berche, Patrick; Ferroni, Agnès

    2007-01-01

    The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

  16. 基于16S rDNA序列的Wolbachia的检测及分型%Detection and type determination of Wolbachia based on 16S rDNA sequences

    Institute of Scientific and Technical Information of China (English)

    曲哲; 丛斌; 褚栋; 董辉

    2009-01-01

    Wolbachia是广泛分布于节肢动物体内的一类共生细菌.采用16S rDNA特异片段的PCR-RFLP方法对烟粉虱Bemisia tabaci (Gennadius)不同生物型及米蛾Corcyra cephalonica (Stainton)共生菌Wolbachia进行了检测与分型分析.基于wsp基因对烟粉虱共生菌B组Wolbachia以及米蛾共生菌Wolbachia进行了系统树分析,并对相应的Wolbachia16S rDNA特异片段进行了克隆、测序以及序列比对.结果表明:16S rDNA的特异片段经NheⅠ酶切后RFLP图谱可有效检测与鉴别Wolbachia.烟粉虱共生菌Wolbachia的16S rDNA特异片段经VspⅠ酶切后可得到预期RFLP图谱,而米蛾共生菌B组Wolbachia (基于wsp序列分析为B组)则产生不同的RFLP图谱.序列分析表明,Nauru型烟粉虱体内B组Wolbachia的16S rDNA片段序列与已知B组Wolbachia对应序列(DQ278884)同源性为100%;米蛾体内B组Wolbachia 16S rDNA特异片段有碱基变异,并存在于VspⅠ识别位点内,这是导致VspⅠ酶切后RFLP图谱不同的原因.结果提示,B组Wolbachia 16S rDNA特异片段经VspⅠ酶切的RFLP图谱存在多态性.本研究结果可为今后Wolbachia的检测与分型提供借鉴.

  17. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    NARCIS (Netherlands)

    Ziesemer, K.A.; Mann, A.E.; Sankaranarayanan, K.; Schroeder, H.; Ozga, A.T.; Brandt, B.W.; Zaura, E.; Waters-Rist, A.; Hoogland, M.; Salazar-García, D.C.; Aldenderfer, M.; Speller, C.; Hendy, J.; Weston, D.A.; MacDonald, S.J.; Thomas, G.H.; Collins, M.J.; Lewis, C.M.; Hofman, C.; Warinner, C.

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gen

  18. Prosthetic joint infection due to Lysobacter thermophilus diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Dhawan, B; Sebastian, S; Malhotra, R; Kapil, A; Gautam, D

    2016-01-01

    We report the first case of prosthetic joint infection caused by Lysobacter thermophilus which was identified by 16S rRNA gene sequencing. Removal of prosthesis followed by antibiotic treatment resulted in good clinical outcome. This case illustrates the use of molecular diagnostics to detect uncommon organisms in suspected prosthetic infections.

  19. Denaturing gradient gel electrophoresis analysis of 16S rDNA V3 fragment of bacteria in transgenic wheat soil%转基因小麦田土壤细菌16S rDNA V3片段的DGGE分析

    Institute of Scientific and Technical Information of China (English)

    丁衬衬; 周艳红; 林凡云; 徐剑宏; 吴季荣; 祭芳; 史建荣

    2011-01-01

    为长期监测转基因作物对土壤微生物菌群变化的影响提供可靠的试验方法.以检测转基因小麦田土壤细菌菌群变化为例,利用细菌特异性引物,扩增得到了土壤细菌16S rDNA V3可变区片段;通过变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE),分析PCR扩增得到的V3可变区片段;根据电泳条带的变化情况评价转基因小麦对土壤环境微生物的安全性.结果表明该方法所提取的DNA,可以不经过纯化直接进行PCR扩增等后续试验;DGGE图谱中条带清晰.该方法可作为长期监测土壤微生物种群变化的试验手段.%This experiment was carried out to establish an effective method for detecting the impact of virus resistant transgenic wheat on its environment bacteria. Variable region fragments of 16S rDNA V3 were amplified by specific and sensitive primers of bacteria, analyzed by denaturing gradient gel electrophoresis ( DGGE) , and applied to evaluate the effect of transgenic wheat on the soil bacterium, according to the change of electrophoretic bands. The results showed that the DNA by this extraction method could be used in PCR without purification. On DGGE profiles, the lighter bands of each lane represented their corresponding dominant microorganisms. The influence of transgenic wheat and non-transgenic wheat on soil microbes by the number and brightness of the bands were obtained, so microorganism population fluctuation could be demonstrated effectively with the method.

  20. Detección de Helicobacter pylori en tejido aórtico humano mediante la amplificación del gen del 16S rDNA Detection of Helicobacter pylori in human aortic tissue through amplification of the 16S rDNA gen

    Directory of Open Access Journals (Sweden)

    María F Escobar

    2005-04-01

    Full Text Available Helicobacter pylori es un patógeno humano reportado de manera frecuente como responsable de afecciones gastrointestinales. En los últimos años, se ha sugerido una asociación causal entre infecciones crónicas por varios patógenos, entre ellos Helicobacter pylori, y la génesis y/o progresión de la aterosclerosis. Aunque se han realizado varios estudios, no hay evidencia contundente de que esta asociación sea verdadera. El propósito de este estudio fue investigar la presencia de Helicobacter pylori en muestras de tejido aórtico de pacientes con diagnóstico clínico de ectasia anulo-aórtica, mediante la amplificación por técnicas de reacción en cadena de la polimerasa (PCR de un fragmento del gen del 16S rDNA de este microorganismo. Se analizaron muestras de ADN de tejido aórtico obtenido de 20 pacientes. Se procesó un fragmento de aorta con lesión aterosclerótica aparente y otro de una región aparentemente sana en cada uno de los pacientes. No se detectaron ácidos nucleicos de Helicobacter pylori en ninguno de los especímenes analizados. Los resultados del estudio sugieren baja o nula asociación entre Helicobacter pylori y enfermedad coronaria en nuestro medio.Helicobacter pylori is a human pathogen, frequently reported as responsible of gastrointestinal affections. During the last years, a causal relationship between chronic infections by several pathogens, among them the Helicobacter pylori, and the genesis and/ or progression of atherosclerosis, has been suggested. Although several studies have been realized, there is no conclusive evidence to assert this association. The purpose of this study was to investigate the presence of Helicobacter pylori in aortic tissue samples from patients with clinical diagnosis of annulo-aortic ectasia, through PCR amplification of a gen fragment of the 16S rDNA of this microorganism. Samples of DNA aortic tissue obtained from 20 patients were analyzed. An aortic fragment with apparent

  1. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    Science.gov (United States)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  2. Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences.

    Science.gov (United States)

    Lee, Simon K Y; Wang, H Z; Law, Sheran H W; Wu, Rudolf S S; Kong, Richard Y C

    2002-05-01

    Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood. However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known. In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them--Vibrio costicola, V. diazotrophicus, V. fluvialis, V. nigripulchritudo, V. proteolyticus, V. salmonicida, V. splendidus and V. tubiashii. The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced. Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios. The three IGS types--IGS(0), IGS(IA) and IGS(Glu)--were the most prevalent forms detected. Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR. The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera. The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study.

  3. Sequence and secondary structure of the mitochondrial 16S ribosomal RNA gene of Ixodes scapularis.

    Science.gov (United States)

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

    The complete DNA sequences and secondary structure of the mitochondrial (mt) 16S ribosomal (r) RNA gene were determined for six Ixodes scapularis adults. There were 44 variable nucleotide positions in the 1252 bp sequence alignment. Most (95%) nucleotide alterations did not affect the integrity of the secondary structure of the gene because they either occurred at unpaired positions or represented compensatory changes that maintained the base pairing in helices. A large proportion (75%) of the intraspecific variation in DNA sequence occurred within Domains I, II and VI of the 16S gene. Therefore, several regions within this gene may be highly informative for studies of the population genetics and phylogeography of I. scapularis, a major vector of pathogens of humans and domestic animals in North America.

  4. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  5. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  6. Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

    Science.gov (United States)

    Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

    2006-05-25

    We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

  7. Who are the active players of the Iberian Margin deep biosphere? Microbial diversity of borehole U1385 through analysis of 16S rDNA and rRNA

    Science.gov (United States)

    Russell, J. A.; Orsi, W.; Edgcomb, V. P.; Biddle, J.

    2013-12-01

    Microbial community structure and activity in marine deep subsurface environments across the globe have been assayed using various molecular biology tools including 16S rDNA sequencing, microarrays, FISH/CARD-FISH, and metagenomics. Many studies involving these techniques are DNA-based. This limits study of microbial function in these environments as DNA does not degrade as quickly as RNA and may lead to misinterpreting relic microbial genes as important for present-day activity. In this study, the diversity of bacteria and archaea from sediments of the Iberian Margin IODP borehole U1385 was analyzed from bulk extracted DNA and RNA at seven different depths ranging from 10 to 123 meters below seafloor (mbsf). Presented data suggests that the picture of microbial diversity obtained from DNA is markedly different from that seen through analysis of RNA. IODP borehole U1385 offers a great comparison to ODP Site 1229, a well characterized borehole on the Peru Margin. Similar sediment depositional history and geochemistry will allow exploration of what represents a 'typical' continental margin sediment microbial community or if microbial endemism is established despite similar conditions. This study represents the first molecular exploration of sediment microbial communities from the Iberian Margin IODP Site U1385.

  8. A renaissance for the pioneering 16S rRNA gene

    Energy Technology Data Exchange (ETDEWEB)

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  9. Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

    Science.gov (United States)

    Akutsu, Tomoko; Motani, Hisako; Watanabe, Ken; Iwase, Hirotaro; Sakurada, Koichi

    2012-05-01

    To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

  10. Identification Lactic acid bacteria isolated from mongolia fermented milk products bases on 16S rDNA sequence analysis and tuf-RFLP technology%利用16S rDNA序列及tuf-RFLP鉴定蒙古国发酵乳中的乳酸菌

    Institute of Scientific and Technical Information of China (English)

    王宏梅; 于洁; 包秋华; 吕嫱; 孟和毕力格; 张和平

    2011-01-01

    One hundred and ten strains of lactic acid bacteria (LAB) isolated from twenty five samples of naturally fermented yoghurt in Dza-vhan of Mongolia were classified by 16S rDNA sequence analysis and tuf- RFLP. They were preliminary identified as Streptococcus thermophilus (41 strains), Lactobacillus helvetkus (40 strains), Lactobarillus delbrueckii subsp. Bulgaricus (11 strains), Lactobadllus fermentum (2 strains), Leuconostoc mesenteroides subsp. Mesenteroides (2 strains), Lactococcus lactis subsp. Lactis (1 strain) and Lactobadllus casei group (12 strains) by 16S rDNA sequence analysis. However, the 16S rDNA sequence of Lactobadllus casei group existed very small difference, tuf-RFLP was used to confirm results given by 16S rDNA sequencing. The tuf- RFLP profile pattern revealed that the twelve strains were accurately identified as Lactobadllus casei.%运用16S rDNA序列分析和tuf-RFLP技术对采于蒙古国扎布汗省的25份发酵乳样中分离出的110株乳酸菌进行鉴定.首先将分离的110株乳酸菌的16S rRNA基因进行扩增,测序并构建系统发育树,初步鉴定为41株嗜热链球菌,40株瑞士乳杆菌,11株德氏乳杆菌保加利亚亚种,2株发酵乳杆菌,1株乳明串珠菌,2株肠膜明串肠膜亚种,1株乳酸乳球乳酸亚种和12株属于干酪族的菌株.由于干酪乳杆菌族的16S rDNA序列差异很小,故采用tuf-RFLP 技术对这12株进行了进一步的验证,通过分离菌株与模式菌株tuf-RFLP图谱的比较分析,结果表明这12株菌均为干酪乳杆菌.

  11. Screening, Isolation and Identification of Probiotic Producing Lactobacillus acidophilus Strains EMBS081 & EMBS082 by 16S rRNA Gene Sequencing.

    Science.gov (United States)

    Chandok, Harshpreet; Shah, Pratik; Akare, Uday Raj; Hindala, Maliram; Bhadoriya, Sneha Singh; Ravi, G V; Sharma, Varsha; Bandaru, Srinivas; Rathore, Pragya; Nayarisseri, Anuraj

    2015-09-01

    16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). Its most important advantage over the traditional biochemical characterization methods is that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel species of Probiotic Lactobacillus acidophilus. The sample was collected from pond water samples of rural and urban areas of Krishna district, Vijayawada, Andhra Pradesh, India. Subsequently, the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The sequence aligned against other species was concluded to be a novel, Probiotic L. acidophilus bacteria, further which were named L. acidophilus strain EMBS081 & EMBS082. After the sequence characterization, the isolate was deposited in GenBank Database, maintained by the National Centre for Biotechnology Information NCBI. The sequence can also be retrieve from EMBL and DDBJ repositories with accession numbers JX255677 and KC150145.

  12. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    Science.gov (United States)

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.

  13. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  14. Comparison of 16S ribosomal RNA genes in Clavibacter michiganensis subspecies with other coryneform bacteria.

    Science.gov (United States)

    Li, X; De Boer, S H

    1995-10-01

    Nearly complete sequences (97-99%) of the 16S rRNA genes were determined for type strains of Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. sepedonicus, and Clavibacter michiganensis subsp. nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that the C. michiganensis subsp. shared relatively high homology with the 16S rRNA gene of Clavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G+C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of the Clavibacter michiganensis subsp., Clavibacter xyli, Arthrobacter globiformis, Arthrobacter simplex, and Frankia sp.; another cluster consisted of members of the corynebacteria-mycobacteria-nocardia (CMN) group of Mycobacteriaceae including Tsukamurella paurometabolum; and Propionibacterium freudenreichii alone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.

  15. Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons.

    Science.gov (United States)

    Kamke, Janine; Taylor, Michael W; Schmitt, Susanne

    2010-04-01

    The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of sponge-associated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high- and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms.

  16. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    Science.gov (United States)

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  17. 16S rDNA测序快速鉴定废水生物处理系统目标细菌%Use of 16S rDNA sequencing for quickly determining target bacteria in biological wastewater treatment system

    Institute of Scientific and Technical Information of China (English)

    李永峰; 任南琪; 杨传平; 陈瑛; 郑国香; 胡立杰

    2005-01-01

    在废水生物处理系统中建立分子生物学技术快速鉴定有关微生物,具有十分重要的意义.以发酵法生物制氢系统的活性污泥分离培养的厌氧发酵细菌为研究对象,采用16S rDNA碱基测序分子生物学技术,通过生物信息学数据库NCBI将DNA序列输入、比对,可以直接鉴定从废水生物处理系统中分离培养的细菌进行种属科的系统发育学地位的判定,从而简化了细菌鉴定程序.通过16S rDNA测序技术,进行了分离产氢细菌的分子生物学鉴定,发现生物制氢厌氧活性污泥中所分离的细菌可能存在的菌属有:Lactobacillus,Clostridium,Klebsiella,Propionibacterium和可能新发现的1个新属Biohydrogenbacterium等5个菌属的菌种,其中新属Biohydrogenbacterium的菌种都以利用碳水化合物生产氢气为其主要特征.16S rDNA测序技术为废水生物处理系统的细菌学研究提供了方便、准确和经济的技术基础.

  18. Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora%东北虎粪细菌区系的16S rRNA基因序列分析

    Institute of Scientific and Technical Information of China (English)

    图雅; 朱伟云; 陆承平

    2005-01-01

    为研究东北虎粪微生物区系建立了东北虎粪细菌的16S rDNA文库.通过EcoRⅠ和HindⅢ分别对阳性克隆进行酶切分析,从东北虎的16S rDNA文库中分别获得了15个具有酶切差异的克隆.BLAST分析结果显示,在15个克隆中,10个克隆与梭菌属成员有97%以上的同源性,其中有6个序列与诺维梭菌A型(Clostridium novyi type A)有99%的同源性,为诺维梭菌A型;4个序列与猪粪细菌RT-18B(Swine manure bacterium RT-18B)有97%的同源性,为消化链球菌属(Peptostreptococcus)成员.其它序列与GenBank中登录的序列同源性低于97%,为5种未培养细菌,其中4种16S rRNA基因序列分别与Clostridium pascui 、破伤风梭菌E88(Clostridium tetani E88)、梭菌(Clostridium sp.)14505及产气荚膜梭菌(Clostridiumperfringens)有94%~95%的相似性.第5种与肉杆菌(Carnobacterium sp.)R-7279株有94%的同源性.

  19. Phylogenetic Analysis of the 16S rDNA of a Strain Isolated from Diseased Larva of Anoplophora glabripennis (Motsch.)%罹病光肩星天牛幼虫分离菌株的16 S rDNA系统发育分析

    Institute of Scientific and Technical Information of China (English)

    邓彩萍; 刘红霞; 闫喜中; 武旭霞; 骆有庆

    2008-01-01

    [Objective] Study on the phylogenetic analysis of the 16S rDNA and insecticidal characteristics of strain BH-1 isolated from diseased larva of Anoplophora glabripennis (Motsch.) [Method] The strain was identified by routine method and inoculated onto healthy Anoplophora glabripennis (Motsch.) for observing insecticidal effect, further 16S DNA was amplified by the specific primers for sequencing and homology analysis. [Result] The mortality of second instar of Anoplophora glabripennis(Motsch.) reached 72.7% 8 d after 1010 cfu/ml BH-1 was inoculated. The homology of 16S DNA se- quences between BH-1 and Serratia marcescens accessed in GenBank reached 99.5%. Combined with the results of routine identification, BH-1 was identi-fied as S. marcescens. [Conclusion] BH-1 could be used for biological control ofAnoplophora glabripenais (Motsch.).

  20. Analysis of soil bacterial community composition by 16S rDNA clone library sampling from transgenic carnation%16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

    Institute of Scientific and Technical Information of China (English)

    白蓝; 赵明文; 贾军伟; 李鹏; 王金斌; 潘爱虎

    2012-01-01

    [目的]通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据.[方法]通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成.[结果]转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异.[结论]通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限.%[Objective] We did this research to analyze the impact on the soil bacterial population of the transgenic carnation, which laid the foundation for the safety assessment of GM carnation. [Methods] Bacterial 16S rDNA gene clone libraries of GM and non-GM carnations were constructed, and the soil bacterial populations of these carnations were compared. [Resultsl The results showed Alphaproteobacteria, Betaproteobacteria, Planctomycetes and Acidobacteria were shared with GM and non-GM carnation. Some differences were found in Actinobacteria, Verrucomicrobia and uncultured bacterium clone. [Conclusion] The results indicated that high bacterial diversity was found in these GM carnation soil bacterial libraries, and the cultivation of genetically modified carnation did not have a significant impact on the soil bacterial community structure.

  1. Molecular identification and detection of moon jellyfish (Aurelia sp.) based on partial sequencing of mitochondrial 16S rDNA and COI%基于mt-16S rDNA和mt-COI基因的海月水母分子生物学鉴定方法和检测技术

    Institute of Scientific and Technical Information of China (English)

    王建艳; 甄毓; 王国善; 米铁柱; 于志刚

    2013-01-01

    以我国近海海域常见的海月水母为目标生物,提取其水母基因组DNA,PCR扩增mt-16S rDNA(650 bp)和mt-COI基因(709 bp)的部分序列,经纯化、克隆、测序后,将获得的序列进行BLASTn分析,筛选目的序列中与其他水母差异较大的区域,设计出8对分别针对海月水母mt-16S rDNA和mt-COI基因序列的特异性引物.经过实验室培养样品和模拟现场样品检测发现,针对mt-16S rDNA的引物AS3和mt-COI的引物AC3具有较好的特异性,可以从海蜇、沙海蜇、霞水母、端鞭水母和海月水母中快捷地检测出目标水母,从而初步建立了海月水母的分子生物学鉴定方法和检测技术.本技术摆脱了水母形态学鉴定方法存在的局限,可准确、快速地对样品进行定性分析.%Taking the moon jellyfish Aurelia sp.commonly found in our coastal sea areas as test object, its genome DNA was extracted, the partial sequences of mt-16S rDNA (650 bp) and mt-COI (709 bp) were PCR-amplified, and, after purification, cloning, and sequencing, the sequences obtained were BLASTn-analyzed.The sequences of greater difference with those of the other jellyfish were chosen, and eight specific primers for the mt-16S rDNA and mt-COI of Aurelia sp.were designed, respectively.The specificity test indicated that the primer AS3 for the mt-16S rDNA and the primer AC3 for the mt-COI were excellent in rapidly detecting the target jellyfish from Rhopilema esculentum, Nemopilema nomurai, Cyanea nozakii, Acromitus sp., and Aurelia sp., and thus, the techniques for the molecular identification and detection of moon jellyfish were preliminarily established , which could get rid of the limitations in classical morphological identification of Aurelia sp., being able to find the Aurelia sp.in the samples more quickly and accurately.

  2. Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR

    OpenAIRE

    Rachman, Cinta; Kabadjova, Petia; Valcheva, Rosica; Prévost, Hervé; Dousset, Xavier

    2004-01-01

    The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in ord...

  3. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    Science.gov (United States)

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  4. PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

    Science.gov (United States)

    Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

    2015-11-01

    Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

  5. 毛竹竹鞭内生细菌的特征及16S rDNA多样性分析%Characteristics of Endophytic Bacteria Isolated from Mosobamboo Rhizome and Their 16S rDNA Diversity

    Institute of Scientific and Technical Information of China (English)

    袁宗胜; 刘芳; 张国防

    2014-01-01

    本研究利用传统的细菌分离方法,结合16S rDNA序列分析对毛竹竹鞭内生细菌的特征和多样性进行了分析.从福建省武夷山、将乐、长汀毛竹竹鞭中分离到34株内生细菌,初步归属于14属,20种.来源于不同地区的毛竹竹鞭内生细菌组成存在较大差异,其优势菌群为产碱杆菌属(Alcaligenes)和葡萄球菌属(Staphylococcus).

  6. Physiological and Biochemical Characteristics of Thermophilic Bacteria and Its 16S rDNA Sequence Analysis%高温菌的生理生化及其16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    李华芝; 李秀艳; 韩波波

    2010-01-01

    利用高温菌耐热嗜热的特性和有机物好氧分解的基本原理,从厨余垃圾处理系统中分离筛选到6株在65℃能产生淀粉酶、脂肪酶、蛋白质酶及纤维素酶的高温高效菌种,并对其生理生化和16S rDNA进行了分析.结果表明:筛选出的6株高温菌,经鉴定都有芽孢,并具有兼氧性;对其进行16S rDNA区段序列测定显示,6株高温菌属于Geobacillus thermoglucosidasius菌种中的不同亚种;分离筛选出的高温菌经过2h的迟缓期后很快进入对数生长期,且持续时间较长,生长速度尤以HB6最快;6株高温菌株全部具有淀粉、纤维素、蛋白质降解活性,5株有脂肪降解活性.可见高温菌具有更高的有机物降解活性和更快的代谢速率.可提高有机物质的降解速率,缩短工艺运行时间,有利于工业化生产,在厨余资源化利用上具有广泛的应用前景.

  7. Genetic Diversity of Potato Scab Pathogens in China Based on 16S rDNA Sequences%中国马铃薯疮痂病病原菌16S rDNA的遗传多样性分析

    Institute of Scientific and Technical Information of China (English)

    张萌; 赵伟全; 于秀梅; 杨文香; 张汀; 刘大群

    2009-01-01

    [目的]利用16S rDNA序列对来自中国8个省(区)的34株马铃薯疮痂病菌和15株参比菌株进行系统发育分析.[方法]采用链霉菌通用引物对34株测试菌株的基因组进行16S rDNA序列扩增,测序后在GenBank中进行BLASTn比对,选取同源性较高的菌株构建系统发育树.[结果]34个测试菌株均为链霉菌属,其中Streptomyces scabies 12株、S.galilacus 8株、S.bobili 6株、S.turgidiscabies 1株、S.acidiscabies1株、S.diastatochzomogenes 2株、S.setonii 1株、S.enissocaesilis 3株.其中S.galilacus、S.bobili和S.enissocaesilis等种均为新发现的疮痂病病原.对各菌株的分布分析发现,山东省的疮痂病菌有4种,内蒙古自治区和陕西省有3种,四川省、河北省和山西省均有2种,黑龙江省为1种.[结论]中国马铃薯疮痂病菌存在明显的遗传多样性,且分布较为复杂.

  8. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Muhammad Naveed

    2014-09-01

    Full Text Available In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ. Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

  9. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

    Science.gov (United States)

    Naveed, Muhammad; Mubeen, Samavia; Khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

  10. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis.

    Science.gov (United States)

    El Gawhary, Somaia; El-Anany, Mervat; Hassan, Reem; Ali, Doaa; El Gameel, El Qassem

    2016-02-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen. © The Author [2015]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Neonatal Meningitis by Multidrug Resistant Elizabethkingia meningosepticum Identified by 16S Ribosomal RNA Gene Sequencing

    Directory of Open Access Journals (Sweden)

    V. V. Shailaja

    2014-01-01

    Full Text Available Clinical and microbiological profile of 9 neonates with meningitis by Elizabethkingia meningosepticum identified by 16S ribosomal gene sequencing was studied. All the clinical isolates were resistant to cephalosporins, aminoglycosides, trimethoprim-sulfamethoxazole, β-lactam combinations, carbapenems and only one isolate was susceptible to ciprofloxacin. All the isolates were susceptible to vancomycin. Six of nine neonates died even after using vancomycin, based on susceptibility results. E. meningosepticum meningitis in neonates results in high mortality rate. Though the organism is susceptible to vancomycin in vitro, its efficacy in vivo is questionable and it is difficult to determine the most appropriate antibiotic for treating E. meningosepticum meningitis in neonates.

  12. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  13. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K.bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

  14. Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species

    Indian Academy of Sciences (India)

    Yogesh S Shouche; Milind S Patole

    2000-12-01

    Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes, Aedes, Anopheles and Culex. The sequence was found to be unusually A + T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished between Aedes and Culex on the one hand, and Anopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.

  15. GENE 16S RRNA SEQUENCE PHYLOGENETIC ANALYSIS OF LYSINE PRODUCERS STRAINS

    Directory of Open Access Journals (Sweden)

    G. S. Andriiash

    2014-12-01

    Full Text Available The phylogenetic relationships of strainsproducers of essential amino acids of aspartate family Brevibacterium sp. UCM Ac-674 (Brevibacterium sp. 90, Brevibacterium sp. IMV Ac-5004 (Brevibacterium sp. 90H, Brevibacterium sp. UCM Ac-675 (Brevibacterium sp. E531, mutant strain Brevibacterium sp. IMV B-7447 from the «Collections strains and lines of plants for food and agricultural biotechnology SO “Institute for Food Biotechnology and Genomics” of National Academy of Sciences of Ukraine were investigated. The affiliation strain Brevibacterium sp. IMV B-7447 to the genus Brevibacterium within the sequences of the genes based on 16S rRNA was confirmed. The dendogram of phylogenetic relationships of studied strains and related strains Brevibacterium from database GenBank was constructed. It was shown that by the criterion of homology gene sequences based on 16S rRNA the investigated strains-producers belong to three phylogenetic groups. It was established that the mutant strain Brevibacterium sp. ІMV B-7447 has no analogues in the database GenBank.

  16. Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing.

    Science.gov (United States)

    Wang, Tao; Kong, Fanrong; Chen, Sharon; Xiao, Meng; Sorrell, Tania; Wang, Xiaoyan; Wang, Shuo; Sintchenko, Vitali

    2011-01-01

    The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

  17. Absence of Helicobacter pylori high tetracycline resistant 16S rDNA AGA926-928TTC genotype in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil

    Directory of Open Access Journals (Sweden)

    Suzuki Rodrigo

    2012-05-01

    EcoRI restriction site. Conclusions H. pylori AGA926-928TTC 16 S rDNA gene substitutions were not found in our population. More research is required to investigate if H. pylori TetR has a different genetic background in our region and if the nucleotide substitutions of the uncultured H. pylori 16 S rRNA partial sequences have biological significance.

  18. Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

    Science.gov (United States)

    Glassing, Angela; Dowd, Scot E; Galandiuk, Susan; Davis, Brian; Jorden, Jeffrey R; Chiodini, Rodrick J

    2015-12-01

    Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

  19. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

    Science.gov (United States)

    Allsopp, M; Visser, E S; du Plessis, J L; Vogel, S W; Allsopp, B A

    1997-08-01

    Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp

  20. [Phylogenetic comparison between Spirulina and Arthrospira based on 16S rRNA and rpoC1 gene].

    Science.gov (United States)

    Wu, Yuemei; Wang, Suying; Dong, Shirui

    2016-02-04

    Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-35 1, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

  1. Partial Sequencing of 16S rRNA Gene of Selected Staphylococcus aureus Isolates and its Antibiotic Resistance

    Directory of Open Access Journals (Sweden)

    Harsi Dewantari Kusumaningrum

    2016-08-01

    Full Text Available The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2,  the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.

  2. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    Science.gov (United States)

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between

  3. Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of common Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to h1 and the h3 haplotype is found only in the coastal waters of China. AG transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

  4. RISSC: a novel database for ribosomal 16S-23S RNA genes spacer regions.

    Science.gov (United States)

    García-Martínez, J; Bescós, I; Rodríguez-Sala, J J; Rodríguez-Valera, F

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S-23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documentation tool for studies on evolution, identification, typing and strain characterization, among others.

  5. discussion on validity of rana maoershanensis based on partial sequence of 16s rrna gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    rana maoershanensis found in mt.maoershan in guangxi,china was reported as a new species in 2007,but there was no molecular data for this frog.the partial sequences (543 bp) of 16s rrna gene from 12 specimens of 3 brown frog species (rana hanluica,r.maoershanensis and r.chensinensis) were analyzed with 17 specimens of 9 species from genbank.the nucleotide sequence divergence between r.maoershanensis and the other brown frog species were 4.5%-6.5%,with 22-30 nucleotide substitutions at this locus.the phylogenetic relationships based on mp,ml,and bayesian inference indicate that the brown frogs from southern china were diverged into three groups (clades a,b and c).r.maoershanensis was clustered together a well-supported subclade (b-l).it is suggested that r.maoershanensis is a valid species.

  6. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    National Research Council Canada - National Science Library

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; Desantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    .... A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species...

  7. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    Science.gov (United States)

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

  8. 基于16S rDNA序列的中国沿海短蛸种群遗传结构%Population genetic structure of Octopus ocellatus in coastal waters of China based on 16S rDNA sequence

    Institute of Scientific and Technical Information of China (English)

    吕振明; 李焕; 吴常文; 樊甄姣; 张建设

    2011-01-01

    采用线粒体基因测序技术,对中国沿海7个短蛸(Octopus ocellatus)群体的遗传结构和变异进行分析,以探讨种群扩散潜力对遗传结构的影响.共测定了100个短蛸样本的.485 bp的16s rDNA序列,经UPGMA聚类分析表明,中国沿海的短蛸群体存在着明显的种群结构.7个群体明显地分化为两大类群:一个类群由北方沿海的大连、烟台、青岛、连云港群体组成,另一个类群南南方沿海的上海、舟山和广东群体组成;两类群间存在14个同定核苷酸碱基的替换.两类群间的遗传分化系数FST和基因流Nm分别达0.878和0.069,并达到显著分化水平(P<0.05).AMOVA检验表明,两类群间的差异有87.76%存在于群体间,而仅有12.24%存在于群体内.两类群间的净遗传距离为0.019,表明可能为中度的种内群体间分化水平.依据分子钟理论推断,两类群的分化时间约为晚更新世冰期.两类群的分化可能与短蛸缺乏浮游幼体生活史和无长距离迁移习性有关,基因流与地理距离间的相关性符合脚踏石模型,进一步证实了这一点.同时短蛸群体的这种分化格局还可能与晚更新世以来中国沿海海平面的反复升降和长江口淡水的阻隔有关.%Genetic structure of populations is greatly influenced by the dispersal ability of marine organisms. It seems intuitive that limited dispersal capability should result in greater genetic differentiation. Octopus ocellatus is a typical cephalopod species which shows limited dispersal potential. In this study, the effect of dispersal capability on population genetic structure was tested on seven populations of O. ocellatus in coastal waters of China,by applying mitochondrial gene sequencing technology. A 485 bp segment of the 16S rDNA gene was sequenced and analyzed in 100 specimens. Results showed that strong genetic structure existed in seven O. ocellatus populations. Two distinct clades with 14 fixed nucleotide base variation

  9. Diversity of 16S-23S rDNA internal transcribed spacer (ITS reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.

    Directory of Open Access Journals (Sweden)

    Andrew P Liguori

    Full Text Available Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

  10. Molecular taxonomy of cupped oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand based on COI, 16S, and 18S rDNA polymorphism.

    Science.gov (United States)

    Klinbunga, S; Khamnamtong, B; Puanglarp, N; Jarayabhand, P; Yoosukh, W; Menasveta, P

    2005-01-01

    Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.

  11. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

    Indian Academy of Sciences (India)

    B. Mahendran; S. K. Ghosh; S. C. Kundu

    2006-04-01

    We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkwormspecies that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

  12. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    Science.gov (United States)

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

  13. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    Science.gov (United States)

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

  14. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    Science.gov (United States)

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

  15. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes.

  16. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  17. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    Directory of Open Access Journals (Sweden)

    Mohammad Bagher Javadi Nobandegani

    2015-01-01

    Full Text Available Phosphate solubilizing bacteria (PSB can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang oil palm field (University Putra Malaysia. Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer in an oil palm field.

  18. DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates

    DEFF Research Database (Denmark)

    Mygind, T; Birkelund, Svend; Christiansen, Gunna

    1998-01-01

    To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified...

  19. Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture.

    Science.gov (United States)

    Garson, Jeremy A; Patel, Poorvi; McDonald, Carl; Ball, Joanne; Rosenberg, Gillian; Tettmar, Kate I; Brailsford, Susan R; Pitt, Tyrone; Tedder, Richard S

    2014-03-01

    Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture. A total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a "universal" probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions. Seven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 "initial-reactive" PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae. These results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing. © 2013 American Association of Blood Banks.

  20. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  1. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

    Directory of Open Access Journals (Sweden)

    Jiazhen Chen

    Full Text Available Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported.The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains.Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72% had intragenomic nucleotide polymorphisms (SNPs in multiple locations, and 13 strains (9.4% had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89 and 100% (19/19 of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities

  2. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    Science.gov (United States)

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  3. Application of 16S rDNA sequencing technique in infantile intestinal microecological research%16SrDNA测序技术在婴儿肠道微生态研究中的应用

    Institute of Scientific and Technical Information of China (English)

    马丽亚; 王辉林; 陈睿; 张敏; 黄艳; 卢光进

    2015-01-01

    目的:探讨16S rDNA测序技术在新生儿、婴儿肠道微生态研究中的应用。方法于生后3天、1月、6月、1岁时收集2例健康婴儿粪便标本共8份,提取细菌总DNA ,以Illumina Hiseq 2000为测序平台,采用新一代高通量16S rDNA宏基因组测序技术对V6可变区测序,并进行生物信息分析(物种分类和丰度分析;多样性分析)。结果8份样品共产生原始测序数据为1027.47 Mbp ,Unique tags序列数量均值为58630,OTU数量63~209;优势菌门为Proteobacteria和Firmicutes ;在科水平,>1%的物种1个月之内2~4种,6月后达7~10种;1号婴儿一直以Enterobacteriaceae占优势,2号婴儿优势菌群包括 Enterobacteriaceae、Lachnospiraceae、Streptococcaceae和Bacteroidaceae;4个时间点的npShannon和Simpson指数分别为1.17、1.29、2.16、2.51和0.43、0.40、0.26、0.14。结论16S rDNA测序技术能满足新生儿、婴儿肠道微生态研究需求;新生儿、婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;从出生到1岁,婴儿肠道菌群结构趋向复杂和多样。%Objective To investigate the application of 16S rDNA sequencing technique in studying intestinal microecology of neonates and infants .Methods Eight fecal samples were collected on 3 d ,1 month ,6 months and 1 year in 2 healthy infants .Total bacterial DNAs were extracted and submitted the high throughout 16S rDNA sequen‐cing on the V6 viable region by using Illumia genome analyzer Hiseq 2000 (101 bp pair‐end sequencing strategy) .The 16S rDNA tags and operational taxonomic units (OTU) were then obtained from the sequences .The bioinformatic a‐nalysis including analysis of taxonomy ,abundance and alpha diversity were performed .Results Total 1 027 .47 Mbp raw data were produced .The mean unique tags number was 58630 .The OTU numbers ranged from 36 to 308 .The bacterial families more than 1% were increased

  4. Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species.

    Science.gov (United States)

    Dey, S; Singh, V P; Kumar, A A; Sharma, B; Srivastava, S K; Singh, Nem

    2007-08-01

    The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

  5. 16S rRNA gene sequencing as a tool to study microbial populations in foods and process environments

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Hansen, Tina Beck; Bahl, Martin Iain

    2015-01-01

    and their role in food safety. During method optimization, we have identified several factors which distort the characterization of microbial populations, including DNA extraction methods, DNA polymerases, and most importantly the analyzed fragment of the 16S rRNA gene. Methods: This study investigated microbial...... reference. Results: Taxonomic assignments and abundances of sequences in the total community and in the Enterobacteriaceae subpopulation were affected by the 16S rRNA gene variable region, DNA extraction methods, and polymerases chosen. However, community compositions were very reproducible when the same......Introduction: Methodological constraints during culturing and biochemical testing have left the true microbiological diversity of foods and process environments unexplored. Culture-independent molecular methods, such as 16S rRNA gene sequencing, may provide deeper insight into microbial communities...

  6. Restrição do 16S-23S DNAr intergênico para avaliação da diversidade de Azospirillum amazonense isolado de Brachiaria spp. Restriction of 16S-23S intergenic rDNA for diversity evaluation of Azospirillum amazonense isolated from different Brachiaria spp.

    Directory of Open Access Journals (Sweden)

    Fábio Bueno dos Reis Junior

    2006-03-01

    Full Text Available O objetivo deste trabalho foi avaliar a diversidade intra-específica de isolados de Azospirillum amazonense e estabelecer a possível influência de diferentes espécies de Brachiaria ssp. e diferentes condições edafoclimáticas. A caracterização da diversidade desses isolados foi conduzida, utilizando-se a análise de restrição da região intergênica 16S-23S DNAr. As estirpes estudadas separaram-se em dois grupos, definidos a 56% de similaridade. As espécies de Brachiaria ssp. influenciaram a diversidade de estirpes. A maioria dos isolados oriundos de B. decumbens e B. brizantha está inserida no primeiro grupo, enquanto os oriundos de B. humidicola concentram-se no segundo grupo.The aim of this work was to study the intra-specific diversity of Azospirillum amazonense isolates and to establish possible influences of different Brachiaria spp. and edaphoclimatic conditions. The characterization of the diversity among the isolates of A. amazonense studied was conducted using restriction analysis of the 16S-23S rDNA intergenic spacer region. The evaluated strains were separated in two groups, defined at 56% of similarity. Brachiaria spp. showed effects on strain diversity. Most part of the isolates from B. decumbens and B. brizantha are inserted in the first group, while B. humidicola isolates concentrate in the second group.

  7. Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing%应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

    Institute of Scientific and Technical Information of China (English)

    王荫榆; 李会荣; 贾士芳; 吴正钧; 郭本恒

    2006-01-01

    以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析.结 果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%.相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter).首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中.

  8. Escherichia coli Vertebral Osteomyelitis Diagnosed According to Broad-range 16S rRNA Gene Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Shibata, Satoshi; Tanizaki, Ryutaro; Watanabe, Koji; Makabe, Kenta; Shoda, Naoki; Kutsuna, Satoshi; Nagamatsu, Maki; Oka, Shinichi; Ohmagari, Norio

    2015-01-01

    Identifying the causative agent of pyogenic osteomyelitis is often challenging, especially when antibiotics are administered before a biopsy. We herein present a case of osteomyelitis in the cervical vertebrae presenting with progressive paralytic symptoms, in which we successfully identified Escherichia coli from a biopsy specimen using broad-range 16S rRNA gene polymerase chain reaction (PCR) even though sensitive antibiotics had been used for more than 50 days before the biopsy. Broad-range 16S rRNA gene PCR is a useful diagnostic method, especially when prebiopsy antibiotics are unavoidably used for a clinically unstable state.

  9. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    Science.gov (United States)

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  10. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.

    Directory of Open Access Journals (Sweden)

    Christian Milani

    Full Text Available Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.

  11. Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data.

    Directory of Open Access Journals (Sweden)

    Manal Helal

    Full Text Available BACKGROUND: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia. METHODS: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization. RESULTS: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52% corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578. CONCLUSION: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra

  12. 16S rRNA gene-based metagenomic analysis reveals differences in bacteria-derived extracellular vesicles in the urine of pregnant and non-pregnant women.

    Science.gov (United States)

    Yoo, Jae Young; Rho, Mina; You, Young-Ah; Kwon, Eun Jin; Kim, Min-Hye; Kym, Sungmin; Jee, Young-Koo; Kim, Yoon-Keun; Kim, Young Ju

    2016-02-05

    Recent evidence has indicated that bacteria-derived extracellular vesicles (EVs) are important for host-microbe communication. The aims of the present study were to evaluate whether bacteria-derived EVs are excreted via the urinary tract and to compare the composition of bacteria-derived EVs in the urine of pregnant and non-pregnant women. Seventy-three non-pregnant and seventy-four pregnant women were enrolled from Dankook University and Ewha Womans University hospitals. DNA was extracted from urine EVs after EV isolation using the differential centrifugation method. 16S ribosomal RNA (16S rRNA) gene sequencing was performed using high-throughput 454 pyrosequencing after amplification of the V1-V3 region of the 16S rDNA. The composition of 13 taxa differed significantly between the pregnant and non-pregnant women. At the genus level, Bacillus spp. EVs were more significantly enriched in the urine of the pregnant women than in that of the non-pregnant women (45.61% vs 0.12%, respectively). However, Pseudomonas spp. EVs were more dominant in non-pregnant women than in pregnant women (13.2% vs 4.09%, respectively). Regarding the compositional difference between pregnant women with normal and preterm delivery, EVs derived from Ureaplasma spp. and the family Veillonellaceae (including Megasphaera spp.) were more abundant in the urine of preterm-delivered women than in that of women with normal deliveries. Taken together, these data showed that Bacillus spp. EVs predominate in the urine of pregnant women, whereas Pseudomonas spp. EVs predominate in the urine of non-pregnant women; this suggests that Bacillus spp. EVs might have an important role in the maintenance of pregnancy.

  13. Globicatella sanguinis bacteraemia identified by partial 16S rRNA gene sequencing

    DEFF Research Database (Denmark)

    Abdul-Redha, Rawaa Jalil; Balslew, Ulla; Christensen, Jens Jørgen;

    2007-01-01

    Globicatella sanguinis is a gram-positive coccus, resembling non-haemolytic streptococci. The organism has been isolated infrequently from normally sterile sites of humans. Three isolates obtained by blood culture could not be identified by Rapid 32 ID Strep, but partial sequencing of the 16S r...

  14. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

    Science.gov (United States)

    Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Background Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. Results The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). Conclusions The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal

  15. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis.

    Science.gov (United States)

    Virtanen, Seppo; Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different

  16. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  17. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications

    DEFF Research Database (Denmark)

    Harrington, C.S.; On, Stephen L.W.

    1999-01-01

    Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0 %, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96.4...

  18. Campylobacter jejuni, an uncommon cause of splenic abscess diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Seng, Piseth; Quenard, Fanny; Menard, Amélie; Heyries, Laurent; Stein, Andreas

    2014-12-01

    Splenic abscess is a rare disease that primarily occurs in patients with splenic trauma, endocarditis, sickle cell anemia, or other diseases that compromise the immune system. This report describes a culture-negative splenic abscess in an immunocompetent patient caused by Campylobacter jejuni, as determined by 16S rRNA gene sequencing.

  19. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  20. 16S rRNA gene sequencing in routine identification of anaerobic bacteria isolated from blood cultures

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Skov, Marianne Nielsine; Knudsen, Elisa;

    2010-01-01

    A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times...

  1. Direct 16S rRNA gene sequencing of polymicrobial culture-negative samples with analysis of mixed chromatograms

    DEFF Research Database (Denmark)

    Hartmeyer, Gitte N; Justesen, Ulrik S

    2010-01-01

    Two cases involving polymicrobial culture-negative samples were investigated by 16S rRNA gene sequencing, with analysis of mixed chromatograms. Fusobacterium necrophorum, Prevotella intermedia and Streptococcus constellatus were identified from pleural fluid in a patient with Lemierre's syndrome...

  2. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    Science.gov (United States)

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  3. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    Science.gov (United States)

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-09-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  4. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    Science.gov (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  5. Diversity of Archaea in Icelandic hot springs based on 16S rRNA and chaperonin genes.

    Science.gov (United States)

    Mirete, Salvador; de Figueras, Carolina G; González-Pastor, Jose E

    2011-07-01

    The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.

  6. Identiifcation of pathogenic microorganism by sequencing 16S rRNA gene%16S rRNA基因序列分析法鉴定病原细菌

    Institute of Scientific and Technical Information of China (English)

    朱飞舟; 陈利玉; 陈汉春

    2013-01-01

    目的:运用16S rRNA 基因序列分析法鉴定14种细菌,为该方法的临床应用奠定基础。方法:提取细菌DNA,采用通用引物PCR扩增16S rRNA 基因片段并测序。将测序结果用Blastn 在线软件在Nucleotide 数据库中进行序列同源性比对,根据序列同源性鉴定病原细菌。结果:12种细菌可以鉴定到“种”,2种细菌可以鉴定到“属”。结论:16S rRNA 基因序列分析是一种有效的病原细菌鉴定方法。%Objective: To identify 14 bacteria by sequencing the 16S rRNA gene and establish the basis for clinical application in the future. Methods: DNA samples of the 14 bacteria were extracted. The 16S rRNA genes were amplified by PCR and sequenced with common primers. The sequences of the 16S rRNA genes were aligned by online software Blastn in nucleotide database. The bacteria were identified according to the homology of their 16S rRNA genes. Results: Twelve bacteria were classified to species, the other 2 bacteria were classified to genus. Conclusion: 16S rRNA gene sequence analysis is useful in identifying pathogenic bacteria.

  7. Progress in Clinical Application of 16S rRNA Gene%16S rRNA基因在临床上的应用进展

    Institute of Scientific and Technical Information of China (English)

    杨祖卿; 尚世强

    2005-01-01

    传统的细菌检测主要依靠血清学、生物化学、细菌形态学及细菌培养等方法进行分类鉴定,但前三者敏感性和特异性不高,后者费时且阳性率低.近10余年来分子生物学技术发展迅速,各种基因方法如DNA杂交、质粒图谱和16S rRNA序列分析等在临床上得到广泛应用.该文就近年来国外16S rRNA在细菌学研究及其应用的一些新进展作一综述.

  8. 16S rRNA基因测序技术在肝脓疡细菌鉴定中的作用%Usefulness of 16S rRNA Gene Sequencing for Identification of Bacteria from Pyogenic Liver Abscess

    Institute of Scientific and Technical Information of China (English)

    宋伦圭

    2014-01-01

    目的:评价16S核糖体RNA (rRNA)基因测序在肝脓疡中细菌鉴定中的应用价值。方法2012年1月-2013年12月间共20例肝脓疡行经皮置管引流的患者,分别行脓液培养,血培养和16S rRNA基因测序。利用454 GS Junior System对脓液基因组DNA行PCR和16S rRNA基因测序。脓液培养,血液培养和16S rRNA 基因测序结果进行分别评价。结果脓液和血液培养阳性的患者分别是9例(45%)和4例(20%)。16S rRNA基因测序细菌鉴定率为90%,明显高于传统的培养方法。结论16S rRNA基因测序方法较传统的培养方法能更准确和有效对肝脓疡进行细菌鉴定。%Background/Aims To evaluate the usefulness of 16S ribosomal RNA (rRNA) gene sequencing for an accurate and better identification of bacteria from pyogenic liver abscess (PLA). Methodology 20 patients with PLA were included who underwent percutaneous catheter drainage, abscess culture, blood culture and 16S rRNA gene sequencing for isolates from January 2012 to December 2013. Genomic DNAs of abscess fluids were subjected to PCR and sequencing of 16S rRNA gene by on a 454 GS Junior System. The results were evaluated between abscess cultures, blood and 16S rRNA gene sequencing for isolates. Results Abscess and blood cultures were positive in 9 (45%) and 4 (20%) patients, respectively. The 16S rRNA gene sequencing showed with 90% identification of bacteria a significantly greater identification than conventional cultured methods. Conclusion This study showed a greater usefulness of 16S rRNA gene sequencing than conventional cultured methods for accurate and better identification of bacteria from PLA.

  9. Bacterial 16S rRNA genes and expression of IL-1β, TNF-α and IgA in prostate tissues%前列腺组织中细菌16S rRNA基因、IL-1β、TNF-α和IgA的表达及意义

    Institute of Scientific and Technical Information of China (English)

    谢辉; 夏鹏; 沈龙捷; 陈洪德; 黄慧聪; 杨亦荣; 吴建波; 何秋香; 朱启建; 陈建欧; 李澄棣

    2010-01-01

    目的 探讨细菌在慢性前列腺炎(CP)中的致病作用.方法 前列腺标本取自2002-2008年192例猝死于非前列腺疾病的器官捐献者,年龄20~38岁.取周围带组织并分两块,一块前列腺组织行病理检查及白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、免疫球蛋白A(IgA)的免疫组化分析;另一块行细菌16S rRNA基因(16S rDNA)PCR分析.结果 33.3%(64/192)的前列腺组织病理呈CP改变.细菌16S rDNA总阳性率为19.8%(38/192),而在CP标本中16S rDNA阳性率为50.0%(32/64),非CP标本中16S rDNA阳性率为4.6%(6/128),CP组16S rDNA阳性率高于非CP组(x2=55.185,P<0.001).IL-1β、TNF-α和IgA的表达在CP组中明显高于非CP标本(P<0.01),且三者表达呈正相关(P<0.01);在64例CP组织标本中,16S rDNA阳性者IL-1β、TNF-α和IgA的表达明显高于16S rDNA阴性者(P<0.01).结论 前列腺组织中细菌16S rDNA、细胞因子和免疫球蛋白A的表达增加和前列腺组织病理炎症改变相关,提示细菌感染可能是CP的重要病因.%Objective To investigate the role of bacteria in the etiology of chronic prostatitis.Methods Complete prostate specimens were obtained at autopsy from 192 organ donors (aged 20 - 38 years old) during 2002 to 2008 who died of non-prostatic diseases.One tissue taken from the peripheral prostatic zone according to McNeal was divided into two pieces.One piece of tissue was taken for routine pathological examinations and immunohistochemical studies of interleukin (IL) -1β, tumor necrosis factor-α (TNF-α)and IgA.Another one was taken for PCR assay to detect the bacterial 16S rRNA genes ( 16S rDNA ).Results Of 192 prostate specimens, 64 (33.3%) had pathological changes of chronic prostatitis and 38 ( 19.8% ) specimens was positive for bacterial 16S rDNA.Positive rates of 16S rDNA in chronic prostatitis and non-prostatitis specimens were 50.0% (32/64) and 4.6% (6/128) respectively ( X2 = 55.185, P <0.001 ).Expressions of IL-1 β, TNF-α and Ig

  10. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    Institute of Scientific and Technical Information of China (English)

    Carlos; W; Nossa; William; E; Oberdorf; Jφrn; A; Aas; Bruce; J; Paster; Todd; Z; DeSantis; Eoin; L; Brodie; Daniel; Malamud; Michael; A; Poles

    2010-01-01

    AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classifica...

  11. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    Science.gov (United States)

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification.

  12. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Science.gov (United States)

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  13. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

    Full Text Available The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX, which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  14. VITCOMIC: visualization tool for taxonomic compositions of microbial communities based on 16S rRNA gene sequences

    Directory of Open Access Journals (Sweden)

    Kurokawa Ken

    2010-06-01

    Full Text Available Abstract Background Understanding the community structure of microbes is typically accomplished by sequencing 16S ribosomal RNA (16S rRNA genes. These community data can be represented by constructing a phylogenetic tree and comparing it with other samples using statistical methods. However, owing to high computational complexity, these methods are insufficient to effectively analyze the millions of sequences produced by new sequencing technologies such as pyrosequencing. Results We introduce a web tool named VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community that can analyze millions of bacterial 16S rRNA gene sequences and calculate the overall taxonomic composition for a microbial community. The 16S rRNA gene sequences of genome-sequenced strains are used as references to identify the nearest relative of each sample sequence. With this information, VITCOMIC plots all sequences in a single figure and indicates relative evolutionary distances. Conclusions VITCOMIC yields a clear representation of the overall taxonomic composition of each sample and facilitates an intuitive understanding of differences in community structure between samples. VITCOMIC is freely available at http://mg.bio.titech.ac.jp/vitcomic/.

  15. [Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

    Science.gov (United States)

    Zhang, Jun-yi; Zhu, Bing-chuan; Xu, Chao; Ding, Xiao; Li, Jun-feng; Zhang, Xue-gong; Lu, Zu-hong

    2015-11-01

    The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes.

  16. 芝麻根际生长素产生菌 SA4的分离与鉴定%Isolation and 16S rDNA identification of IAA-producing strain SA4 from rhizosphere of sesame

    Institute of Scientific and Technical Information of China (English)

    陆娟; 苏利梅; 胡名扬; 唐俊

    2015-01-01

    利用含色氨酸的鉴定培养基从芝麻根际筛选出7株 IAA 产生菌,其中菌株 SA4产 IAA 的能力最强。促生实验结果表明,菌株 SA4明显具有促进黄瓜和玉米种子主根生长的能力,使它们的主根根长和根重增加为对照组的2~3倍。分子鉴定结果表明菌株 SA4属于革兰氏阴性的假单胞菌属。%Seven strains producing IAA were isolated from rhizosphere of sesame by the differential medium containing L-tryptophan, and strain SA4 was the best one producing IAA. The strain SA4 had obvious ability to promote the axial root growth of cucumber seedlings and maize seedlings. It made the axial root twice to three times as long as the control group, and also made the root fresh weight twice to three times as much as the control group. Strain SA4 was Gram negative one, and it was identified as Pseudomonas sp. based on the analyse of its 16S rDNA.

  17. PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli

    Science.gov (United States)

    Miyazaki, Kentaro; Sato, Mitsuharu; Tsukuda, Miyuki

    2017-01-01

    We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the “central pseudoknot” and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A–916U and betaproteobacterial 19C–916G) and the non-native 19A–916G pair retained function, whereas the non-native 19C–916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A–916U or 19C–916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts.

  18. Mutational robustness of 16S ribosomal RNA, shown by experimental horizontal gene transfer in Escherichia coli

    OpenAIRE

    Kitahara, Kei; Yasutake, Yoshiaki; Miyazaki, Kentaro

    2012-01-01

    The bacterial ribosome consists of three rRNA molecules and 57 proteins and plays a crucial role in translating mRNA-encoded information into proteins. Because of the ribosome’s structural and mechanistic complexity, it is believed that each ribosomal component coevolves to maintain its function. Unlike 5S rRNA, 16S and 23S rRNAs appear to lack mutational robustness, because they form the structural core of the ribosome. However, using Escherichia coli Δ7 (null mutant of operons) as a host, w...

  19. Phylogenetic assessment of a new cypermethrin- degrading bacterium Gordonia sp.D2 based on 16S rDNA, gyrB and GyrB sequences%一株氯氰菊酯降解菌16S rDNA,gyrB和GyrB的系统发育分析

    Institute of Scientific and Technical Information of China (English)

    张久刚; 闫艳春

    2008-01-01

    A cypermethrin - degrading bacterium named as Gordonia sp. D2 was isolated from activated sludge of a wastewater treatment biore-actor. At 30 ℃ and pH 7.0, the removal rate of 100 mg cypennethrin L-1 was approximately 52.3% in mineral salts medium within 7.5 days, and extra carbon source addition could enhance the biodegradation. The isolate was identified as Gordonia genus through physio - bio- chemical identification combined with the phylogenetic analyses based on 16S rRNA, gyrB nucleotide and GyrB amino acid sequences. 16S rRNA sequences of strain D2 showed the highest similarity to G. amicalis DSM44461 and G. hydrophobica DSM44015T, with the same value of 98.1% ,meanwhile, D2 shows the highest similarity to G. hydrophobica JCA10086 with the value of 86.8% and 91.1% based on gyrB and GyrB. Three phylogenetic dendrograms were made, and results showed that 16S rRNA gene is suit for identifying the isolate to the genus level, gyrB gene and GyrB amino acid sequences are more adapted for phylogenetic analysis.%从农药厂污水处理池中分离得到一株氯氰菊酯降解菌,在30℃,pH 7.0的条件下,无机盐培养基中100mg/L的氯氰菊酯,经过7.5天,能够降解大约52.3%,外加碳源能够明显提高其降解性能.生理生化实验结合16S rDNA,gyrB和GyrB的系统发育分析,将其归为Gordonia菌属.在16S rDNA水平上,其与G.amicalis DSM44461和G.hydrophobica DSM44015的相似值最高,为98.1%;而在gyrB和GyrB水平上,其与G.hydrophobica JCM10086的相似值最高,分别为86.8%和91.1%.通过对所构建的系统发育树进行评估,表明16S rDNA序列适用于将分离菌株鉴定到属的水平上,而gyrB和GyrB更适用于在属内种的水平上进行系统发育的分析.

  20. Preparation of Metagenome from Soil and Analysis on the Sequence of a Bacteria 16S rDNA by Culture-independant Method%土壤宏基因组的提取及基于免培养技术分析细菌16S rDNA

    Institute of Scientific and Technical Information of China (English)

    方光伟; 洪雪梅; 蔡丽希; 彭锟; 林毅

    2005-01-01

    采用CTAB-SDS-冻融法和玻璃粉吸附法提取土壤宏基因组(metagenome),经琼脂糖凝胶电泳后用回收试剂盒对其进行纯化.以细菌16S rDNA通用引物从宏基因组中扩增出V8和V9两个高变区,回收纯化后与克隆载体pMD18-T连接,转化大肠杆菌DH5α,随机挑取一个阳性克隆菌进行序列测定,经BLAST分析表明该序列所代表的是一个不动杆菌属(Acinetobacter)细菌.本研究结果为分析土壤微生物种群结构和直接从土壤中分离功能基因奠定了基础.

  1. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  2. Phylogenetic relationship of 16 Oedipodidae species (Insecta: Orthoptera) based on the 16S rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    HUI-MENG LU; YUAN HUANG

    2006-01-01

    The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phyloge netic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A+T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

  3. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute...... for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  4. Detection and quantification of Dehalogenimonas and "Dehalococcoides" populations via PCR-based protocols targeting 16S rRNA genes.

    Science.gov (United States)

    Yan, Jun; Rash, Brian A; Rainey, Fred A; Moe, William M

    2009-12-01

    Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to "Dehalococcoides" but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

  5. Deep sequencing of 16S rRNA gene to efficiently assess bacterial richness and the rare biosphere in soils

    OpenAIRE

    Terrat, Sébastien; Dequiedt, Samuel; Bachar, Dipankar; Christen, Richard; Mougel, Christophe; Lelievre, Mélanie; Maron, Pierre-Alain; Plassart, Pierre; Wincker, Patrick; Cruaud, C; Jolivet, Claudy; Arrouays, Dominique; Bispo, Antonio; Lemanceau, Philippe; Ranjard, Lionel

    2012-01-01

    Soil is one of the most important reservoirs of microbiological diversity on our planet and, above all, one of the last bastions of such biodiversity. Since two decades, numerous molecular tools have been developed to assess accurately this huge diversity directly from soil DNA. In this context, 16S rRNA gene is widely used to study microbial community taxonomic diversity, as it can be easily amplified by PCR, and large databases are available relating obtained sequences to bacteria...

  6. Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene

    OpenAIRE

    2012-01-01

    Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from...

  7. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  8. Clinical Fusobacterium mortiferum Isolates Cluster with Undifferentiated Clostridium rectum Species Based on 16S rRNA Gene Phylogenetic Analysis.

    Science.gov (United States)

    Lee, Yangsoon; Eun, Chang Soo; Han, Dong Soo

    2016-05-01

    The most commonly encountered clinical Fusobacterium species are F. nucleatum and F. necrophorum; other Fusobacteria, such as F. mortiferum and F. varium, have occasionally been isolated from human specimens. Clostridium rectum is a gram-positive species characterized as a straight bacillus with oval sub-terminal spores. The close 16S rRNA gene sequence relationship of C. rectum with the genus Fusobacterium is unexpected given their very different phenotypic characteristics. Between 2014 and 2015, a total of 19 Fusobacterium isolates were recovered from the colonic tissue of 10 patients at a university hospital. All isolates were identified based on 16S rRNA gene sequencing. The phylogenetic relationship among these isolates was estimated using the neighbor-joining method and the Molecular Evolutionary Genetic Analysis (MEGA) version 6. Based on phylogenetic analysis, the F. mortiferum isolates clustered into two groups - F. mortiferum DSM 19809 (group I) and F. mortiferum ATCC 25557 (group II) - even though they are of the same species. Furthermore, the F. mortiferum DSM 19809 (group I) showed a close phylogenetic relationship with C. rectum, even though C. rectum is classified as a gram-positive spore-producing bacillus. C. rectum is clearly unrelated to the genus Clostridium as it shows highest 16S rRNA gene sequence similarity with species from the genus Fusobacterium Therefore, additional methods such as Gram staining and other biochemical methods should be performed for Fusobacterium identification.

  9. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  10. Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes.

    Science.gov (United States)

    Costa, Pedro; Amaro, Ana; Ferreira, Ana S; Machado, Diana; Albuquerque, Teresa; Couto, Isabel; Botelho, Ana; Viveiros, Miguel; Inácio, João

    2014-12-01

    Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows the assessment of the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows us to infer the species of the isolate as M. tuberculosis (if all RDs are present), Mycobacterium caprae (if only RD1 and RD4 are present) and Mycobacterium bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CI(P95%) 0.929-1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostic framework of veterinary diagnostic laboratories, with a particular focus for reference laboratories.

  11. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  12. Experimental study of 16S rRNA gene sequence analysis for identification of atypical bacteria%16S rRNA基因序列分析鉴定非典型细菌的实验研究

    Institute of Scientific and Technical Information of China (English)

    沈亚娟; 夏云

    2013-01-01

    目的 建立以16S rRNA基因序列分析为基础的细菌鉴定方法,并初步将其应用于临床常规细菌的鉴定.方法 选择临床微生物实验室不能准确鉴定的细菌,以16S rRNA为靶序列,在两端保守区设计引物,PCR反应扩增目的 片段,测序后与数据库中已知细菌的16S rRNA序列进行序列比对.结果 13株菌中,有11株与数据库中的已知16S rRNA序列相似性达99.0%以上,成功鉴定到种的水平.结论 16S rRNA基因序列分析的方法可快速、准确地鉴定不典型菌株,可作为细菌常规鉴定的补充方法.%Objective To establish an approach for bacteria identification based on 16S rRNA gene sequence analysis ,and apply it to clinical routine bacteria identification initially .Methods Bacteria which can not be accurately identified in clinical microbiologi -cal laboratory were selected .16S rRNA served as target sequence ,primers were designed to match the conserved region at both ends and target fragments were amplified by means of PCR reaction .After sequencing ,their sequences were compared with 16S rRNA sequence of known bacteria in the database .Results Among 13 strains ,11 strains showed sequence similarity of 99 .0% with 16S rRNA sequence of known bacteria in the database ,and were successfully identified to the species level .Conclusion 16S rRNA gene sequence analysis can be applied to identify atypical bacteria quickly and accurately ,and serve as a supplementary method of routine bacteria identification .

  13. 大白菜软腐病菌16S rDNA序列比对鉴定及杀菌剂对其生物活性测定试验%16S rDNA Sequence Compare of Erwinia carotovora subsp, carotovora and Bioactivity of Fungicide against Chinese Cabbage Soft Rot

    Institute of Scientific and Technical Information of China (English)

    陈亮; 刘君丽; 司乃国

    2011-01-01

    [Aims] Erwinia carotovora subsp. Carotovora is an important plant disease having effect on the normal growth of Chinese cabbage, which seriously results in decreasing crop's yield and quality. Chemical control is an effective messure to control the disease. In order to clarify the controlling effect of strobilurins and antibiotics on Erwinia carotovora subsp. Carotovora, the research was carried out. [Methods] E. Carotovora subsp. Carotovora separated from the leaf of rotten Chinese cabbage was identified using method of sequence comparison to 16S rDNA, and then bioactivity of the fungicides mentioned above was tested using E. Carotovora subsp. Carotovora as target. [Results] The results indicated that there was great bioactivity difference between different fungicides for E. Carotovora subsp. Carotovora, the results of in vitro and in vivo are different for the same fungicide. [Conclusions] Due to the bioactivity difference between in vitro and in vivo, these two testing methods, in vivo and in vitro should be evaluated together to obtain accurate evaluation.%[目的]大白菜软腐病是影响白菜正常生长的重要病害,严重影响白菜的产量和品质.化学防治是控制大白菜软腐病的重要措施.为了进一步明确甲氧基丙烯酸酯类和抗生素类药剂对大白菜软腐病的防治效果,开展试验研究.[方法]从腐烂大白菜叶片分离得到大白菜软腐病菌(Erwinia carotovora subsp.carotovora),采用16S rDNA序列比对的方法对其进行鉴定.将其作为防治对象,测试杀菌剂的生物活性.[结果]不同药剂对大白菜软腐病菌的生物活性差异很大,同一药剂在离体试验和活体试验中的结果也并不相同.[结论]由于药剂的生物活性测试结果在离体和活体试验中表现出差异性,需要将2种试验方法相结合,才能对药剂生物活性进行准确评价.

  14. 16S rRNA基因检测对新生儿败血症诊断价值的Meta分析%Meta-analysis of value of 16S rRNA gene detection in diagnosis of neonatal sepsis

    Institute of Scientific and Technical Information of China (English)

    王远明; 杜惠容; 陈恒; 张辉

    2012-01-01

    目的 16S rRNA 基因检测是诊断新生儿败血症的方法之一,但是尚无研究综合评价其诊断价值,本研究拟系统评价16S rRNA基因检测在新生儿败血症中的诊断价值.方法 在Cochrane图书馆、Medline、Embase、Science Direct、中国期刊全文数据库、万方、维普等数据库中查找利用16S rRNA基因检测诊断新生儿败血症的文献.QUADAS工具评价纳入文献的质量,Meta-Disc 1.4 软件检验异质性并根据其结果选择相应效应模型计算纳入研究的合并敏感性、特异性等指标,绘制汇总受试者工作特征(SROC)曲线,综合评价16S rRNA基因检测的诊断价值.结果共有8篇文献共计2 543例新生儿纳入本次研究,Meta分析显示16S rRNA基因检测对新生儿败血症的合并诊断价值分别为:敏感度为0.93,特异度为0.97,阳性似然比为25.12,阴性似然比为0.08,诊断比值比为323.40.SROC曲线下面积为0.99.结论 16S rRNA基因检测中对新生儿败血症具有较高的诊断价值,可作为诊断新生儿败血症重要工具.%Objective 16S Rrna gene detection is one of the methods for the diagnosis of neonatal sepsis, but there is not any study evaluating its diagnostic value comprehensively. So this study aims to evaluate the diagnostic value of 16S Rrna gene detection in neonatal sepsis comprehensively. Methods Literature on diagnosis of neonatal sepsis using the 16S Rrna gene detection was searched in Cochrane Library, Medline, Embase, Science Direct, CNKI, Wanfang, VIP and other databases. QUADAS tool was used to evaluate the quality of literature; Meta-Disc 1.4 software was used to test heterogeneity, based on which the relevant effect model was selected to calculate the combined sensitivity, specificity and other indicators of the literature, the summary receiver operating characteristic (SROC) curve was drawn and the diagnostic value of 16S Rrna gene detection was evaluated comprehensively. Results A total of 8 pieces of

  15. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    Science.gov (United States)

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

  16. Establishment of the pathogen 16 s rDNA detection method in dog and monkey common-ly used animal for cancer research%肿瘤研究常用动物犬和猴病原菌16srDNA检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    时伟红; 郑志红

    2015-01-01

    目的:建立肿瘤研究常用动物犬和猴病原菌的16s rDNA PCR检测方法。方法:提取犬病原菌布鲁杆菌、空肠弯曲杆菌、小肠结肠炎耶尔森菌和猴病原菌志贺氏菌DNA,经过通用引物扩增,PCR产物为病原菌16s rDNA全长,经纯化后直接测序。利用BLAST软件从GenBank数据库中搜索相关菌株的序列进行序列比对和同源性分析,确定病原菌的种属。结果:测序结果与数据库中搜索到的相关菌株的序列进行序列比对显示,4种病原菌测序分别与布鲁杆菌、空肠弯曲杆菌、小肠结肠炎耶尔森菌和志贺氏菌序列一致。结论:利用16s rDNA PCR方法可快速准确地检测肿瘤研究常用动物犬和猴病原菌,对保证肿瘤研究准确性有重要意义。%Objective:To establish the pathogen detection method in dog and monkey commonly used animal for cancer research. Methods:The DNA of Brucella spp,Campylobaceter jejuni,Yersinia enterocolitica and Shigella spp confirmed by conventional methods were extracted. The full length of 16s rDNA was amplified with a set of universal primers. The PCR products were purified and sequenced directly. The related sequences were obtained from the Gen-Bank database using BLAST search software and the species of the pathogen were identified according to the sequence analysis and homology comparison of the 16s rDNA sequences. Results:The 16s rDNA sequences of the four pathogen were 100% concordance with the known standard nueleotide sequences of Brucella spp,Campylobaceter jejuni, Yersinia enterocolitica and Shigella spp. Conclusion:The PCR method based on 16s rDNA was reliable and could i-dentify dog and monkey the pathogen rapidly and accuracy.

  17. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    OpenAIRE

    Eisen, J. A.; Smith, S W; Cavanaugh, Colleen Marie

    1992-01-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and b...

  18. Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria

    NARCIS (Netherlands)

    Salzman, NH; de Jong, H; Paterson, Y; Harmsen, HJM; Welling, GW; Bos, NA

    2002-01-01

    Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of

  19. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Science.gov (United States)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  20. The evaluation of an identification algorithm for Mycobacterium species using the 16S rRNA coding gene and rpoB

    Directory of Open Access Journals (Sweden)

    Yuko Kazumi

    2012-01-01

    Conclusions: The 16S rRNA gene identification is a rapid and prevalent method but still has some limitations. Therefore, the stepwise combination of rpoB with 16S rRNA gene analysis is an effective system for the identification of Mycobacterium species.

  1. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed;

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  2. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    Science.gov (United States)

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  3. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette; Søborg, Ditte Andreasen; Al-Soud, Waleed Abu

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  4. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

  5. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  6. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    Science.gov (United States)

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  7. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    Science.gov (United States)

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  8. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

    Directory of Open Access Journals (Sweden)

    Patrick D. Schloss

    2016-03-01

    Full Text Available Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  9. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  10. Phenotypic characterization and 16S rDNA identification of culturable non-obligate halophilic bacterial communities from a hypersaline lake, La Sal del Rey, in extreme South Texas (USA)

    Science.gov (United States)

    2012-01-01

    Background La Sal del Rey ("the King's Salt") is one of several naturally-occurring salt lakes in Hidalgo County, Texas and is part of the Lower Rio Grande Valley National Wildlife Refuge. The research objective was to isolate and characterize halophilic microorganisms from La Sal del Rey. Water samples were collected from the lake and a small creek that feeds into the lake. Soil samples were collected from land adjacent to the water sample locations. Sample salinity was determined using a refractometer. Samples were diluted and cultured on a synthetic saline medium to grow halophilic bacteria. The density of halophiles was estimated by viable plate counts. A collection of isolates was selected, gram-stained, tested for catalase, and characterized using API 20E® test strips. Isolates were putatively identified by sequencing the 16S rDNA. Carbon source utilization by the microbial community from each sample site was examined using EcoPlate™ assays and the carbon utilization total activity of the community was determined. Results Results showed that salinity ranged from 4 parts per thousand (ppt) at the lake water source to 420 ppt in water samples taken just along the lake shore. The density of halophilic bacteria in water samples ranged from 1.2 × 102 - 5.2 × 103 colony forming units per ml (cfu ml-1) whereas the density in soil samples ranged from 4.0 × 105 - 2.5 × 106 colony forming units per gram (cfu g-1). In general, as salinity increased the density of the bacterial community decreased. Microbial communities from water and soil samples were able to utilize 12 - 31 carbon substrates. The greatest number of substrates utilized was by water-borne communities compared to soil-based communities, especially at lower salinities. The majority of bacteria isolated were gram-negative, catalase-positive, rods. Biochemical profiles constructed from API 20E® test strips showed that bacterial isolates from low-salinity water samples (4 ppt) showed the greatest

  11. Molecular Identification of 4 Strains of Aeromonas Based on 16 S rDNA Sequence%基于16SrDNA序列的4株气单胞菌属细菌的分子鉴定

    Institute of Scientific and Technical Information of China (English)

    谭凤霞

    2012-01-01

    16SrDNA sequences were amplified and sequenced for four Aeromonas bacteria strains isolated from the aquacul-ture water. The homologous sequences were searched and retrieved using BLAST, then sequences were aligned by MegAlign sofeware using Jotun Hein, ClustalV and ClustalW methods. Phylogenetic trees were constructed by Mega 4.0 software using Neighbor-joining (N-J), minimum evolution (ME), maximum parsimony (MP) and UPGMA methods. Sequences analysis showed that there are 99.2% (Jotun Hein) similarity between Aeromonas sp. T3 16 S rDNA and DQ817542.1, DQ817645.1 and HM127065.1; Aeromonas sp. T4 and DQ816364.1, HM77846.1 and HM778618.1 have 97.9% (Jotun Hein) similarity; Aeromonas sp. T5 and GQ205446.1 have 99.4% (Jotun Hein) similarity; Aeromonas sp. T6 and GQ205446.1 have 99.6% (Jotun Hein) similarity. Similar results were supported by the other two alignment methods. Phylogenetic trees constructed by the four methods showed similar results, and the taxonomic status for the four isolated strains could be investigated. Results showed that Aeromonas sp. T3 and DQ817542.1, Aeromonas sp. T4 and HM778618.1, Aeromonas sp. T5 and CQ232759.1, Aeromonas sp. T6 and GQ205446.1 had the closest relationship, respectively.%对分离自养殖水环境的4株气单胞菌属(A eromonas)细菌的16 S rDNA序列进行PCR扩增并测定其核酸序列,通过BLAST软件在GenBank中查找同源序列,应用MegAlign软件中的Jotun Hein、ClustalV以及ClustalW 3种方法进行序列差异和同源性分析,分别使用Mega 4.0软件中的邻接法(NJ)、最小进化法(ME)、最大简约法(MP)、非加权组平均法(UPGMA)构建系统发育树.由Jotun Hein法可知,Aeromonas sp.T3与序列DQ817542.1、DQ817645.1以及HM127065.1相似度最高,均为99.2%,Aeromonas sp.T4与序列DQ816364.1、HM77846.1以及HM778618.1相似度最高,均为97.9%,A eromonas sp.T5与序列GQ205446.1相似度最高,均为99.4%,Aeromonas sp.T6与序列GQ205446.1相似度最高,为99.6%;其

  12. Identification of Bacillus strains isolated from Yacai by 16S rRNA gene sequencing%利用16S rRNA序列鉴定分离自芽菜中的芽孢杆菌

    Institute of Scientific and Technical Information of China (English)

    张良; 吴华昌; 邓静; 李萍萍; 肖辰; 沈芳

    2012-01-01

    从宜宾芽菜中分离优势菌群,选取4株芽孢杆菌,分别为B1、B2、B3、B4.对4株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立芽孢杆菌属的系统发育树.结合细菌形态学生理生化特性鉴定结果,结果表明菌株B1、B3为枯草芽孢杆菌,菌株B2为解淀粉芽孢杆菌,菌株B4为乙酰微小杆菌.%Four dominant Bacillus strains named Bl, B2, B3 and B4 were isolated from Yacai (a kind of Yibin pickles in China). The 16S rRNA genes of these strains were amplified in vitro and sequenced. Then a phylogenetic tree was constructed by multiple alignments of their sequences with other 16S rRNA gene sequences of Bacillus. According to 16S rRNA gene analysis combined with morphological, physiological and biochemical characteristics, B1 and B3 were identified as Bacillus subtilis, B2 was Bacillus amyloliquefaciens and B4 was Exiguobacterium acetylicum.

  13. DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes.

    Science.gov (United States)

    Feng, Yanwei; Li, Qi; Kong, Lingfeng; Zheng, Xiaodong

    2011-01-01

    DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller's classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.

  14. IDENTIFICATION OF Staphylococcus sp. STRAINS ISOLATED FROM POSITIVE WIDAL BLOOD BASED ON 16S rRNA GENE SEQUENCES

    Directory of Open Access Journals (Sweden)

    Sri Darmawati

    2015-12-01

    Full Text Available The purpose of this study is to identify 8 strains of Staphylococcus genus members isolated from positive Widal blood (4 strains of Staphylococcus saprophyticus, 1 strain of Staphylococcus warneri, 2 strains of Staphylococcus hominis, 1 strain of Staphylococcus xylosus and 1 strain of Staphylococcus capitis based on 16S rRNA gene sequences. The methods used in this study are conducting PCR of 16S rRNA gene, cloning genes using T-Vector pMD20 which is transformed to E. coli DH5α, sequencing. The results show that four strains (BA 47.4, BA 19.2, KD 29.5 and TG 09.1 are identified as Stap. Saprophyticus strains of Stap. saprophyticus members of ATCC 15305T (99.01-100% similarity. The strain of TG 01.3 is identified as Stap. Warneri strain of TG 01.3 of Stap. Warneri members of ATCC 27836T (99.74-99.93% similarity, 2strains (KT 29.2 and KD 35.1 are identified as of Stap. hominis members of DSM 20328T (99.4-99.67% similarity. The strain of KT 30.5 is not identified

  15. Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, M.; Khanna, S. [NIIT Univ, Neemrana (India). Dept. of Biotechnology & Bioinformation

    2010-04-15

    In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available. Coal-tar-contaminated soil was collected, which consisted of 122-122.5 mg g{sup -1} total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (I) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center {alpha}-subunit) common to all PAH dioxygenase enzymes and (iii) {beta}-subunit of dioxygenase. Phylotypes related to Proteobacteria ({Alpha}-, {Epsilon}- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of alpha-subunit and beta-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type (2Fe2S) cluster binding site suggested that these gene fragments encode for {alpha}-subunit of dioxygenase gene. Sequencing of the cloned libraries representing {alpha}-subunit gene fragments (Rf1) and beta-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil.

  16. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

    Science.gov (United States)

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-01-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species. PMID:25313278

  17. 16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea, Echinodermata)

    Institute of Scientific and Technical Information of China (English)

    MA Haiyan; JIANG Guoliang; WU Zhiqiang; WANG Xin

    2009-01-01

    Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.

  18. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    Science.gov (United States)

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

  19. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    Science.gov (United States)

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  20. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    Directory of Open Access Journals (Sweden)

    Ilias Lagkouvardos

    2017-01-01

    Full Text Available The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  1. Microbial diversity in Diaphorina citri( Homoptera: Psyllidae) estimated by 16S rDNA analysis using DGGE and RFLP%基于16S rDNA序列的柑桔木虱体内共生菌多样性研究

    Institute of Scientific and Technical Information of China (English)

    殷幼平; 刘婷婷; 田圣超; 胡修峰; 吴东; 王中康

    2011-01-01

    昆虫消化道内是一个复杂的微生态系统,有大量的微生物存在.这些微生物对寄主发育、营养吸收和防御方面都起着重要的作用.本文利用基于16S rRNA基因的PCR-RFLP指纹图谱法和变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)的方法对柑桔黄龙病虫-媒柑桔木虱Diaphorina citri体内细菌菌群多样性进行了研究.经PCR-RFLP分析显示31条序列与变形菌门的假单胞菌科、肠杆菌科、黄单胞菌科、伯克氏菌科、立克次氏菌科和根瘤菌科细菌具有较高同源性.柑桔木虱内共生细菌的优势菌群依次为合胞体共生菌(syncytiumendosymbiont)(同源性99%,5条序列,分离频率31%)、Candidatus Carsonella ruddii和Mycetocyte内共生菌(同源性98%,5条序列,分离频率31%)以及亚洲韧皮杆菌(Candidatus Liberibacter asiaticus)和内共生菌Wolbachia.对柑桔木虱内生细菌16S rDNA V3区序列的PCR-DGGE分析,条带相似性的UPMAGA聚类分析表明,采自九里香Murraya paniculata的柑桔木虱内生细菌大多聚为一支,而来自柑桔的聚在另一支,说明寄主差异对柑桔木虱内生细菌菌群构成的影响大于地理位置的影响.将PCR-DGGE条带测序,序列经GenBank序列比对发现柑桔木虱内生细菌主要属于变形菌门假单胞菌科、立克次氏菌科、肠杆菌科、黄单胞菌科以及厚壁菌门链球菌科和芽孢杆菌科.合胞体共生菌(条带3-4)因其稳定存在于木虱体内且不随柑桔木虱寄主和地理位置的改变而变化,可能是柑桔木虱体内的优势共生菌;内共生细菌Wolbachia也在柑桔木虱内稳定存在,表明我国柑桔木虱感染Wolbachia是普遍现象.PCR-RFLP和PCR-DGGE两种方法相结合较好地反映了柑桔木虱内生细菌菌群的多样性,而且均显示出柑桔木虱内的合胞体共生菌是主要的优势菌群.

  2. The study of 16S rRNA methylase genes in Enterobacter cloacae%庆大霉素耐药阴沟肠杆菌16S rRNA甲基化酶基因的研究

    Institute of Scientific and Technical Information of China (English)

    李玉珍; 吴燕峰; 李红玉; 杨银梅

    2011-01-01

    目的 了解庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的分布情况.方法 筛选临床分离的庆大霉素耐药的阴沟肠杆菌30株.采用聚合酶链反应(PCR)方法扩增16S rRNA甲基化酶五种相关耐药基因armA、rmtA、rmtB、rmtC和rmtD,PCR产物进行电泳分析和测序,抗菌药物的药物敏感性试验采用纸片扩散法进行,Whonet 5.4软件进行数据分析.结果 30株庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的检出率为56.7%(17/30),其中30.0%(9/30)检出armA基因、26.7%(8/30)检出rmtB基因,未检出rmtA、rmtC和rmtD基因.30株庆大霉素耐药阴沟肠杆菌对妥布霉素、阿米卡星耐药率分别为93.3%,83.3%;对其他抗菌药物除亚胺培南较敏感外,其余抗菌药物耐药率大于60.0%.结论 庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因主要是armA和rmtB基因,碳青霉烯类对该菌有较好体外抗菌活性.%Objective To investigate the distribution of 16S rRNA methylase genes in gentamicin-resistant Enterobacter cloacae.Method 30 strains of clinically isolated Enterobacter cloacae were collected.Five 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC and rmtD, were detected by polymerase chain reaction (PCR).The PCR products were confirmed by electrophoresis analysis and sequencing.The Antimicrobial Susceptibility Testing was conducted by disk diffusion method.The data were analyzed by Whonet 5.4 software.Result The relevance ratio of 16S rRNA methylase genes were 56.7% (17/30) including armA genes 30.0% (9/30) and rmtB genes 26.7% (8/30).None of the isolates carried rmtA, rmtC and rmtD genes.The resistence rate of Enterobacter cloacae to amikacin and tobramycin were 93.3% and 83.3% respectively.While being sensitive to imipenem, the strains were resistant to many other antimicrobial drugs with the resistance rate over 60.0%.Conclusion 16S rRNA methylase genes in Enterobacter cloacae are mainly armA and rmtB genes

  3. Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species.

    Science.gov (United States)

    Wang, Min; Cao, Boyang; Yu, Qunfang; Liu, Lei; Gao, Qili; Wang, Lei; Feng, Lu

    2008-11-01

    The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types-ITS(none) (without tRNA genes), ITS(glu) [with a tRNA(Glu (UUC)) gene], and ITS(ile+ala) [with tRNA(Ile (GAU)) and tRNA(Ala (UGC)) genes]-were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS(glu) and ITS(ile+ala). The presence of ITS(none) in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS(none), from 0.967 to 1.000 for ITS(glu), and from 0.968 to 1.000 for ITS(ile+ala). Interspecies sequence identities range from 0.775 to 0.989 for ITS(none), from 0.798 to 0.997 for ITS(glu), and from 0.712 to 0.985 for ITS(ile+ala). Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.

  4. Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis.

    Science.gov (United States)

    de la Haba, Rafael R; Arahal, David R; Márquez, M Carmen; Ventosa, Antonio

    2010-04-01

    A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

  5. Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location.

    Science.gov (United States)

    Cabral-de-Mello, Diogo C; Cabrero, Josefa; López-León, María Dolores; Camacho, Juan Pedro M

    2011-07-01

    We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.

  6. Genetic variability of Echinococcus granulosus based on the mitochondrial 16S ribosomal RNA gene.

    Science.gov (United States)

    Wang, Ning; Wang, Jiahai; Hu, Dandan; Zhong, Xiuqin; Jiang, Zhongrong; Yang, Aiguo; Deng, Shijin; Guo, Li; Tsering, Dawa; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2015-06-01

    Echinococcus granulosus is the etiological agent of cystic echinococcosis, a major zoonotic disease of both humans and animals. In this study, we assessed genetic variability and genetic structure of E. granulosus in the Tibet plateau, using the complete mitochondrial 16 S ribosomal RNA gene for the first time. We collected and sequenced 62 isolates of E. granulosus from 3 populations in the Tibet plateau. A BLAST analysis indicated that 61 isolates belonged to E. granulosus sensu stricto (genotypes G1-G3), while one isolate belonged to E. canadensis (genotype G6). We detected 16 haplotypes with a haplotype network revealing a star-like expansion, with the most common haplotype occupying the center of the network. Haplotype diversity and nucleotide diversity were low, while negative values were observed for Tajima's D and Fu's Fs. AMOVA results and Fst values revealed that the three geographic populations were not genetically differentiated. Our results suggest that a population bottleneck or population expansion has occurred in the past, and that this explains the low genetic variability of E. granulosus in the Tibet Plateau.

  7. 机采血小板细菌16s rRNA基因检测的临床研究%Clinical study of bacterial 16s rRNA gene detection in apheresis platelets

    Institute of Scientific and Technical Information of China (English)

    周火根; 池妤

    2009-01-01

    目的 探讨快速、可靠的检测机采血小板(PLT)细菌污染的新方法.方法 用聚合酶链反应(PCR)扩增1 308份献血员机采PLT 16s rRNA基因,以乙型肝炎病毒-脱氧核糖核酸(HBV 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倍倍比稀释法进行该方法的敏感性检测.结果 检测1308份献血员机采PLT,其中7份16s rRNA基因为阳性,阳性率为0.54%(7/1 308).而与HBV 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 × 10~4cfu/L大肠埃希菌.结论 献血员机采PLT板细菌污染的阳性率为0.54%;利用PCR检测献血员机采PLT细菌16s rRNA基因的方法具有特异性、快速性和敏感性高等特点.%Objective To explore a rapid and reliable method for the detection of bacterial contamination in the apheresis platelets. Methods The fragment of bacterial 16s rRNA gene,taken from apheresis platelets of 1 308 blood donors, was amplified by polymerase chain reaction(PCR). Using HBV DNA, Candida albicans and human genome DNA as controls,the specificity was tested. The sensitivity test was performed by the method of 10 gradual dilution. Results 7 of 1 308 alpheresis platelets samples showed 16s rRNA gene positive and the bacterial contamination ratio of apheresis platelets was 0.54%. The cross-reaction with the HBV DNA, Candida albicans and human genome DNA was negative. PCR exhibited sensitivity as low as 1.5 × 10_4 cfu/L Escherichia coli. Conclusions The bacterial detection method of 16s rRNA gene offers the adventages of high specificity, sensitivity and rapidity.

  8. Analysis of the genotypes among different strains of common Mycobacteria based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences%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

    Institute of Scientific and Technical Information of China (English)

    黄至澄; 徐黔宁; 闫李侠; 陈保文; 王国治

    2011-01-01

    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个型别,18株偶发分枝杆菌分为6个型别,17株耻垢分枝杆菌分为4个型别,8株戈登分枝杆菌分为3个型别,9株龟分枝杆菌龟亚种分为3个型别,15株堪萨斯分枝杆菌分为2个型别,17株产鼻疽分枝杆菌分为1个型别;而16S-23S rRNA ITS可依次将上述分枝杆菌分为3个、15个、7个、3个、4个、3个、5个型别.结论 16S rRNA G ene分析和16S-23S rRNA ITS分析均是分枝杆菌基因型分析的可靠方法,此外,16S-23SrRNA ITS的种内多态性高于16S rRNA Gene.

  9. 16S rRNA甲基化酶在产KPC酶肺炎克雷伯菌中的分布%The distribution of 16S rRNA methylase genes in KPC-producing Klebsiella pneumoniae strains

    Institute of Scientific and Technical Information of China (English)

    陆理英; 张伟丽; 杨青; 周华; 俞云松

    2009-01-01

    目的 了解产肺炎克雷伯菌碳青霉烯酶-2型(KPC-2)肺炎克雷伯菌中16S rRNA甲基化酶基因的分布.方法 收集37株产KPC-2肺炎克雷伯菌,使用琼脂稀释法测定其对阿米卡星、庆大霉素和奈替米星的最小抑菌浓度(MIC),PCR扩增6种16S rRNA甲基化酶基因:armA、rmtA、rmtB、rmtC、rmtD和npmA.结果 产KPC-2肺炎克雷伯菌对阿米卡星、庆大霉素和奈替米星的耐药率均为97.3%(MIC50≥1024μg/mL),其中8株检出arms基因,25株检出rmtB基因,同时检出armA和rmtB基因的有4株,未检测到rmtA、rmtC、rmtD和npmA阳性菌株.16S rRNA甲基化酶基因总阳性率为78.4%(29/37).结论 16S rRNA甲基化酶基因armA和rmtB在产KPC-2肺炎克雷伯菌中广泛分布.%Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

  10. Identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp.

    Science.gov (United States)

    Fernández-No, I C; Böhme, K; Caamaño-Antelo, S; Barros-Velázquez, J; Calo-Mata, P

    2015-04-01

    The main goal of this work was the identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp. that may be useful for typing purposes. These species include, among others, Bacillus cereus, an important pathogenic species involved in food poisoning, and Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus, which are causative agents of food spoilage described as responsible for foodborne disease outbreaks. With this purpose in mind, 52 Bacillus strains isolated from culture collections and fresh and processed food were considered. SNP type "Y" at sites 212 and 476 appeared in the majority of B. licheniformis studied strains. SNP type "R" at site 278 was detected in many strains of the B. subtilis/Bacillus amyloliquefaciens group, while polymorphism "Y" at site 173 was characteristic of the majority of strains of B. cereus/Bacillus thuringiensis group. The analysis of SNPs provided more intra-specific information than phylogenetic analysis in the cases of B. cereus and B. subtilis. Moreover, this study describes novel SNPs that should be considered when designing 16S rRNA-based primers and probes for multiplex-PCR, Real-Time PCR and microarray systems for foodborne Bacillus spp.

  11. 病犬大肠杆菌16S rRNA甲基化酶基因检测%Molecular Detection of 16S rRNA Methylase Genes Among Escherichia coli Strains Isolated from Diseased Dogs

    Institute of Scientific and Technical Information of China (English)

    陈玉霞; 李德喜; 杜向党; 潘玉善; 刘建华; 苑丽; 张素梅

    2011-01-01

    为了解郑州市发病犬大肠杆菌对氨基糖苷类药物高度耐药的16S rRNA甲基化酶流行情况,主要研究测定了分离自河南郑州市宠物医院发病犬的123株大肠杆菌对氨基糖苷类代表药物阿米卡星的敏感性;分别设计6种16S rRNA甲基化酶基因特异性引物,对耐药分离株进行16S rRNA甲基化酶基因PCR扩增检测.检测结果显示,在发病犬大肠杆菌中仅检测到armA和rmtB,其检出率分别为3.25%(4/123)和38.2%(47/123).其中,有3.25%(4/123)的大肠杆菌可同时检测到armA和rmtB.这些结果提示,此地区发病犬大肠杆菌16S rRNA甲基化酶基因以rmtB为主.病犬细菌一旦携带这些耐药基因,可导致对氨基糖苷类药物高度耐药,应引起重视.%In order to investigate the epidemiology of 16S rRNA methylases which mediated the high level resistance to aminoglycosides among Escherichia coli strains isolated from diseased dogs in zhengzhou city of He' nan Province. 123 E. coli strains were isolated from clinic samples in diseased dogs. Antibacterial susceptibility determinations were performed and six types of 16S rRNA methylase genes were detected by PCR. Of six types of 16S rRNA methylase genes, only armA and rmtB were detected and the positive rates among these E. coli strains were 3.25% (4/123)and 38.2% (47/123), respectively. The concurrence rate of armA and rmtB genes in E. coli strains in diseased dogs was 3.25%. These results suggested 16S rRNA methylases among E. coli strains isolated from diseased dogs were prevalent in Zhengzhou City of He' nan Province, with rmtB dominant. One the pathogens in dogs carried these resistant genes, which could result in the high level resistance to aminoglycosides. So, this serious situation should cause concern.

  12. Phylogenetic studies of Chinese labeonine fishes (Teleostei:Cyprinidae) based on the mitochondrial 16S rRNA gene

    Institute of Scientific and Technical Information of China (English)

    LI Junbing; WANG Xuzhen; HE Shunping; CHEN Yiyu

    2005-01-01

    The mitochondrial 16S ribosomal RNA gene is sequenced from 24 ingroups taxa, including 18 species from Labeoninae grouped in 13 genera. Phylogenetic analyses are subjected to neighbor joining, maximum parsimony, maximum likelihood and Bayesian analyses. Phylogenetic analysis indicates that Labeoninae is basically a monophyletic assemblage and can be divided into 2 major clades: one comprising the genera Cirrhinus, Crossocheilus and Garra; and the other consisting of the genera Labeo, Sinilabeo, Osteochilus, Pseudoorossocheilus, Parasinilabeo, Ptychidio, Semilabeo, Pseudogyricheilus, Rectori and Discogobio. According to our present analysis,the features such as the presence of the adhesive disc on the chin and the pharyngeal teeth in 2 rows used in the traditional taxonomy of Labeoninae provide scarce information for phylogeny of labeonine fishes.

  13. Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

    Science.gov (United States)

    Laurent, Frederic J.; Provost, Frederique; Boiron, Patrick

    1999-01-01

    Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples. PMID:9854071

  14. Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes

    DEFF Research Database (Denmark)

    Holmsgaard, Peter N.; Sørensen, Søren J.; Hansen, Lars Hestbjerg

    2013-01-01

    The use of amplicon pyrosequencing makes it possible to produce thousands of sequences of the same gene at relatively low costs. Here we show that it is possible to simultaneously sequence the 16S rRNA gene, IncP-1 trfA gene and mercury reductase gene (merA) as a way for screening the diversity o...

  15. Anaerobic, non-sporulating, Gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing.

    Science.gov (United States)

    Lau, Susanna K P; Woo, Patrick C Y; Fung, Ami M Y; Chan, King-man; Woo, Gibson K S; Yuen, Kwok-yung

    2004-12-01

    Owing to the difficulties in identifying anaerobic, non-sporulating, Gram-positive bacilli in clinical microbiology laboratories, the epidemiology and clinical spectrum of disease of many of these bacteria have been poorly understood. The application of 16S rRNA gene sequencing in characterizing bacteraemia due to anaerobic, non-sporulating Gram-positive bacilli during a 4-year period is described. The first case of Olsenella uli bacteraemia, in a patient with acute cholangitis, is also reported. Among 165 blood culture isolates of anaerobic, Gram-positive bacilli, 75 were identified as Propionibacterium acnes by phenotypic tests and 21 as members of other anaerobic, non-sporulating Gram-positive bacilli by 16S rRNA gene sequencing. Of these 96 isolates, 16 (17 %) were associated with cases of clinically significant bacteraemia, among which 10 (63 %) were caused by Eggerthella, four (25 %) by Lactobacillus and one (6 %) by each of Eubacterium tenue and O. uli. Five of the 10 Eggerthella isolates were Eggerthella lenta, whereas the other five belonged to two novel Eggerthella species, with Eggerthella hongkongensis being almost as prevalent as Eggerthella lenta. Underlying disease in the gastrointestinal tract, isolation of Eggerthella and Lactobacillus, and monomicrobial bacteraemia were associated with clinically significant bacteraemia, whereas isolation of P. acnes and polymicrobial bacteraemia were associated with pseudobacteraemia. Most patients with clinically significant bacteraemia had underlying diseases, with diseases in the gastrointestinal tract being most common. The overall mortality rate was 31 %. Immunocompromised patients with clinically significant bacteraemia due to anaerobic, non-sporulating, Gram-positive bacilli other than P. acnes should be treated with appropriate antibiotics. The unexpected frequency of isolation of Eggerthella from blood cultures and its association with clinically significant disease suggest that this genus is probably of

  16. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Loreto Abusleme

    2014-04-01

    Full Text Available Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the

  17. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report.

    Science.gov (United States)

    Lee, Sin Hang

    2016-01-01

    Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain.

  18. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing.

    Science.gov (United States)

    Abusleme, Loreto; Hong, Bo-Young; Dupuy, Amanda K; Strausbaugh, Linda D; Diaz, Patricia I

    2014-01-01

    The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve

  19. Analysis of microbiota associated with peri-implantitis using 16S rRNA gene clone library

    Directory of Open Access Journals (Sweden)

    Tatsuro Koyanagi

    2010-05-01

    Full Text Available Background: Peri-implantitis (PI is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified. Objective: The purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences. Design: Three subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique. Results: A total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46% were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions. Conclusions: The biofilm in PI showed a more complex microbiota when compared to periodontitis and

  20. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

    Science.gov (United States)

    Callejón, R; Halajian, A; de Rojas, M; Marrugal, A; Guevara, D; Cutillas, C

    2012-05-25

    Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis).

  1. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

    Directory of Open Access Journals (Sweden)

    Jan S Suchodolski

    Full Text Available BACKGROUND: Canine idiopathic inflammatory bowel disease (IBD is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6 and dogs with moderate IBD (n = 7 or severe IBD (n = 7 as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001. Proportions of Fusobacteria (p = 0.010, Bacteroidaceae (p = 0.015, Prevotellaceae (p = 0.022, and Clostridiales (p = 0.019 were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044 and Acinetobacter (p = 0.040, were either more abundant or more frequently identified in IBD dogs. CONCLUSIONS/SIGNIFICANCE: In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

  2. Report of Nocardia species isolated from soil by 16S rRNA gene sequencing method in Isfahan, Iran

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    samane bourbour

    2016-03-01

    Full Text Available Introduction: The genus Nocardia belongs to aerobic Actinomycetes. They are a large group of soil-dwelling bacteria that are distributed worldwide. Using various key conventional and molecular diagnostic tests, several Nocardia isolates were identified, characterized and distinguished. Materials and methods: In our study, soil isolation method was Slip-buried type and DNA extraction was obtained through Microwave oven method and Nocardia asteroides DSM 43757-type strain as standard was tested to approve the above method. Following this study, Polymerase Chain Reaction was carried out using universal primers. Results: Phenotypic and molecular data analysis, particularly 16S rRNA gene sequencing, provided evidence on Nocardia cyriacigeorgica KC577151, Nocardia asteroides KC577152, Nocardia cummidelens KC577153, Nocardia asteroides KC577154, Nocardia asteroides KC577155, Nocardia coubleae KC577156 involvement in Iran soil in Isfahan and these isolates were eventually registered in NCBI gene bank. Discussion and conclusion: In this research, six Nocardia species were isolated and identified some of which isolated species were novel in Iran soil in Isfahan, at the time of isolation and detection. To identify more species of Nocardia in subsequent studies, proliferation and sequencing of other genes of the bacteria such as hsp65, ITS, secA, rpoB and sod can be applied. 

  3. 食品中弓形菌16S rRNA特异性扩增检测方法的建立%Development of a specific amplification method of Arcobacter 16S rRNA gene in foods

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。%One pair of primers were used to amplify 16S rRNA sequence of Arcobacter,and a PCR method for detection of Arcobacter was developed with optimization of PCR amplification conditions.PCR products of 3 different Arcobacter type strains were sequenced and compared with 16S rRNA genes of Arcobacter published on NCBI.It's proved that the homology between the PCR products and 16S rRNA genes of Arcobacter published were all above 99%.3 different type strains of Arcobacter produced a 1202 bp amplified band,while all of the other 19 different bacteria didn't produced any band.55 food samples were detected whether Arcobacter present or not by the PCR method following enrichment in Johnson-Murano broth,and 6 samples were detected positive for this microorganism,namely with the positive ratio of 10.9%.The result suggested that the method developed was specific,easy to operate and timesaving,and could be used for rapid detection of Arcobacter in foods.

  4. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    Science.gov (United States)

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-01-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

  5. Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

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    Carmen Fernández-Molina

    2008-10-01

    Full Text Available OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG. La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5% fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU. Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5% were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

  6. 16S rRNA、16S-23S rRNA基因测序分析检测主要血流感染病原菌比较%Comparison of the role of 16S rRNA and 16S-23S rRNA gene sequence-based identification of bacteria in bloodsteam infection

    Institute of Scientific and Technical Information of China (English)

    金中淦; 葛平; 徐蓉; 陈蓉; 宣瑛; 刘学杰; 王庆忠

    2012-01-01

    目的 比较细菌16S rRNA、16S-23S rRNA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16S rRNA、16S-23S rRNA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,16S rRNA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-23S rRNA基因可作为血流感染细菌检测较好的分子靶标.

  7. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  8. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  9. Description of an unusual Neisseria meningitidis isolate containing and expressing Neisseria gonorrhoeae-Specific 16S rRNA gene sequences.

    Science.gov (United States)

    Walcher, Marion; Skvoretz, Rhonda; Montgomery-Fullerton, Megan; Jonas, Vivian; Brentano, Steve

    2013-10-01

    An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.

  10. Molecular origin analysis of Shigella boydii Str. hn03 isolated from chicken based on 16S rRNA,gyrB,grpE and recA genes%16S rRNA基因联合gyrB、grpE、recA基因对鸡源鲍氏志贺菌hn03株进行分子起源分析

    Institute of Scientific and Technical Information of China (English)

    杨霞; 陈陆; 赵军; 王新卫; 常洪涛; 刘红英; 姚慧霞; 王川庆

    2011-01-01

    The sequences of 16S rDNA, gyrB, grpE, recA of Shigella were analysed for the farther identification of newly found Shigella boydii strain isolated from chicken and the evolutionary position of the new strain was determined. Part of 16S rRNA and other three genes were amplified by polymerase chain reaction (PCR) from one identified Shigella boydii strains isolated from chicken and the PCR products were cloned and sequenced. These sequences obtained in this study were compared with the related sequneces reported in GenBank by software of DNA analyses. The similarity were gained and phylogenetic tree were constructed. Further evolutionary relationships among chicken Shigella boydii and other related strains were examined based on sequences. Shigella boydii isolated from chicken had the nearest homogeneity with Shigella boydii isolated from human and their homogeneity is 99.6 %-100 %. Shigella boydii isolated from chicken was in a small cluster with Shigella boydii isolated from human in four phylogenetic trees,and almost all Shigella strains are distributed among E. coli strains. Genes recA and gyrB shown a greater evolutionary divergence than 16S rRNA genes. Sequence analysis of 16S rDNA and gyrB,grpE, recA may be a reliable, rapid and low costs way for identification of Shigella strains. The homogeneity and the phylogenetic tree constructed by these four genes above revealed that the Shigella strain isolated from chicken may be a new species or subspecies of Shigella boydii.%为了从基因水平鉴定鸡源鲍氏志贺菌,分别根据GenBank中登陆的相应序列针对16srRNA、gyrB、grpE、recA4基因设计4对引物,用PCR扩增出鸡源鲍氏志贺菌4基因的部分片段并进行了克隆测序。将所获序列与GenBank中同源性较高的相关序列相比较,计算种间相似性,并构建志贺菌的系统发育树,对分离株进行分类与鉴定。研究发现,从4基因的序列分析结果总体上可知鸡

  11. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    Science.gov (United States)

    Eisen, J A; Smith, S W; Cavanaugh, C M

    1992-05-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.

  12. Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC-IGS and 16S-23S ITS as markers: investigation of horizontal gene transfer.

    Science.gov (United States)

    Piccin-Santos, Viviane; Brandão, Marcelo Mendes; Bittencourt-Oliveira, Maria Do Carmo

    2014-08-01

    Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S-23S ITS) and phycocyanin intergenic spacer (PC-IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC-IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC-IGS and 16S-23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC-IGS. Thus, PC-IGS is a suitable marker, along with 16S-23S ITS for phylogenetic studies of cyanobacteria.

  13. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

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    Mudassar Anisoddin Kazi

    Full Text Available The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2 phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  14. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

    Science.gov (United States)

    Kazi, Mudassar Anisoddin; Reddy, C R K; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  15. High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coli clinical isolates from a Chinese teaching hospital

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    Zhang Xue-qing

    2010-06-01

    Full Text Available Abstract Background Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern. Methods Between January 2006 and July 2008, 680 distinct Escherichia coli clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum β-lactamase (ESBL genes, including armA and rmtB, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the E. coli isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE. Results Among the 680 E. coli isolates, 357 (52.5%, 346 (50.9% and 44 (6.5% isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the rmtB gene and only one harboring armA. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680 and 84.1% (37/44, respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four rmtB-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the armA gene contained ISCR1 and tnpU, the latter a putative transposase gene,. Another putative transposase gene, tnpD, was located within a region downstream of armA. Moreover, a

  16. Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes

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    Mu Peng

    2015-09-01

    Full Text Available Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

  17. First microbiota assessments of children's paddling pool waters evaluated using 16S rRNA gene-based metagenome analysis.

    Science.gov (United States)

    Sawabe, Toko; Suda, Wataru; Ohshima, Kenshiro; Hattori, Masahira; Sawabe, Tomoo

    2016-01-01

    Insufficient chloric sterilization of children's paddling pool waters increases the risk of diarrheal illness. Therefore, we investigated the microbiota changes after children use pools. First, we applied 16S rRNA gene-based metagenome analysis to understand the dynamics of microbiota in pool water, especially with respect to the bio-contamination by potential pathogens. Proteobacteria were major taxa detected in every pool water sample after children spent time in the pool. In more detail, Gammaproteobacteria comprised the dominant class, which was followed by Betaproteobacteria. Five phyla, Bacteroidetes, Firmicutes, Actinobacteria and Deinococcus-Thermus phyla were minor groups. The pool water microbiota are likely to be a consortium of intestinal and skin microbiota from humans. Interestingly, the ratio of Gammaproteobacteria and Betaproteobacteria differed according to the age of the children who used the pool, which means the pool water was additionally contaminated by soil microbiota as a result of the children's behavior. Furthermore, potential pathogens, such as Campylobacter spp., Comamonas testosteroni and Burkholderia pseudomallei, were also found. Considering the standard plate counts, the abundances of these human pathogens are unlikely to be a sufficiently infectious dose. We suggest the importance of sanitary measures in paddling pool waters to reduce bio-contamination from both humans and the environment.

  18. Phylogenetic analysis of the order Pleuronectiformes (Teleostei based on sequences of 12S and 16S mitochondrial genes

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    Marisa F.C. Azevedo

    2008-01-01

    Full Text Available The fish order Pleuronectiformes, composed of 14 families, has two suborders: Psettodoidei (with one family and Pleuronectoidei (with thirteen families. The relationships among families of Pleuronectoidei and among the genera of their families have extensively been debated and a consensus has not yet been reached. In the present study, partial sequences of the 12S and 16S mitochondrial rRNA genes were obtained from 19 species belonging to the families Achiridae, Bothidae, Cynoglossidae, Paralichthyidae, Pleuronectidae, Scophthalmidae, and Soleidae. Additional sequences of 42 pleuronectiform species were obtained from GenBank. Phylogenetic analyses were conducted by the methods of maximum-parsimony, maximum-likelihood and Bayesian inference. Our results corroborate the monophyletic status of all families, excluding Paralichthyidae. In the family Achiridae, the genus Catathyridium (freshwater was the sister group of Trinectes (saltwater, and Hypoclinemus (freshwater was the sister group of Achirus (saltwater. Assuming that the putative ancestor of achirids lived in saltwater, it is suggested that the freshwater habitats in South America were colonized independently by different achirid lineages.

  19. Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes.

    Science.gov (United States)

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-09-24

    Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

  20. Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene.

    Science.gov (United States)

    Bowman, Jeff S; Rasmussen, Simon; Blom, Nikolaj; Deming, Jody W; Rysgaard, Søren; Sicheritz-Ponten, Thomas

    2012-01-01

    Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon's indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student's t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.

  1. Decoupled distance-decay patterns between dsrA and 16S rRNA genes among salt marsh sulfate-reducing bacteria.

    Science.gov (United States)

    Angermeyer, Angus; Crosby, Sarah C; Huber, Julie A

    2016-01-01

    In many habitats, microorganisms exhibit significant distance-decay patterns as determined by analysis of the 16S rRNA gene and various other genetic elements. However, there have been few studies that examine how the similarities of both taxonomic and functional genes co-vary over geographic distance within a group of ecologically related microbes. Here, we determined the biogeographic patterns of the functional dissimilatory sulfite reductase gene (dsrA) and the 16S rRNA gene in sulfate-reducing bacterial communities of US East Coast salt marsh sediments. Distance-decay, ordination and statistical analyses revealed that the distribution of 16S rRNA genes is strongly influenced by geographic distance and environmental factors, whereas the dsrA gene is not. Together, our results indicate that 16S rRNA genes are likely dispersal limited and under environmental selection, whereas dsrA genes appear randomly distributed and not selected for by any expected environmental variables. Selection, drift, dispersal and mutation are all factors that may help explain the decoupled biogeographic patterns for the two genes. These data suggest that both the taxonomic and functional elements of microbial communities should be considered in future studies of microbial biogeography to aid in our understanding of the diversity, distribution and function of microorganisms in the environment.

  2. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections.

    Science.gov (United States)

    Fenollar, Florence; Roux, Véronique; Stein, Andréas; Drancourt, Michel; Raoult, Didier

    2006-03-01

    The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection.

  3. Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study

    Directory of Open Access Journals (Sweden)

    Guillaume eMinard

    2014-05-01

    Full Text Available The Asian tiger mosquito Aedes (Stegomya albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated however. Some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimise the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 98,520 sequences obtained. Ae. albopictus is known to harbour two Wolbachia strains, wAlbA and wAlbB, and quantitative PCR was used to estimate the relative densities, i.e. the bacteria-to-host gene ratios, of the strains in individual mosquitoes. Relative densities were between 6.25 × 100.01 and 5.47 × 100.1 for wAlbA and between 2.03 × 100.1 and 1.4 × 101 for wAlbB. Apart from Wolbachia, a total of 32 bacterial taxa were identified at the genus level using the different in method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from

  4. Identification of polybacterial communities in patients with postoperative, posttraumatic, and endogenous endophthalmitis through 16S rRNA gene libraries.

    Science.gov (United States)

    Jayasudha, Rajagopalaboopathi; Narendran, Venkatapathy; Manikandan, Palanisamy; Prabagaran, Solai Ramatchandirane

    2014-05-01

    Endophthalmitis is a potential vision-threatening complication following surgical procedures (postoperative endophthalmitis [POE]), trauma (posttraumatic endophthalmitis [PTE]), and bacteremic seeding of the eye from a distant infection site (endogenous endophthalmitis [EE]). Several studies have revealed the polybacterial characteristics of endophthalmitis, which make the administration of antibiotics to treat the disease challenging. However, until now, the polybacterial communities of POE, PTE, and EE have not been precisely studied. Hence, the present study was designed to identify the bacterial community of endophthalmitis through 16S rRNA gene libraries. Of the 40 intraocular samples tested, 30 libraries were constructed with bacterial nested-PCR-positive samples. The obtained recombinant clones were screened through amplified rRNA gene restriction analysis (ARDRA) to identify unique clones. The multiple types of ARDRA patterns (P=0.345) and diverse bacterial sequences (P=0.277) within the libraries revealed the polybacterial nature of infection in POE, PTE, and EE. Moreover, to the best of our knowledge, this is the first report on polybacterial infection in EE. Gram-positive bacteria, including Bacillus spp. (n=19), Streptococcus spp. (n=18), Staphylococcus spp. (n=6), Exiguobacterium spp. (n=3), Gemella spp. (n=2), Enterococcus spp. (n=2), a Lysinibacillus sp. (n=1), a Clostridium sp. (n=1), and a Nocardia sp. (n=1), and Gram-negative bacteria, including Serratia spp. (n=18), Pseudomonas spp. (n=10), Enterobacter spp. (n=8), Acinetobacter spp. (n=3), Pantoea spp. (n=3), a Haemophilus sp. (n=1), and a Massilia sp. (n=1), were identified. Interestingly, among them, 10 bacterial species were not previously reported to be associated with endophthalmitis or other ocular infections. Besides, the presence of 4 unidentifiable clones suggests the possibility of novel organisms that might cause eye infections. Therefore, it is recommended that, in addition to the

  5. 16S rRNA Gene Sequence Analysis and Fermentation Products Identification of Amycolatopsis sp.FJNU1011%Amycolatopsis sp. FJNU1011的16S rRNA基因序列分析和发酵产物鉴定

    Institute of Scientific and Technical Information of China (English)

    李力; 刘小玲; 黄连琴; 黄建忠

    2013-01-01

    从土壤中分离1株具有抗革兰氏阳性菌和抗肿瘤活性的放线菌,其16S rRNA的序列聚类分析结果表明该菌可能是拟无枝酸菌属中的一个新种,定命为Amycolatopsis sp.FJNU1011.通过液相色谱质谱联用仪对其发酵提取物进行分析,发现不仅含有5种抗肿瘤作用kigamicins的组分,并且含有新的kigamicins结构类似物.%A strain isolated from soil has the inhibitory activities against G + bacterial and tumor. The sequence analysis of 16S rRNA gene showed that the strain is a species of amycolatop sis, named Amycolatopsis sp. FJNU1011. With the analysis by HPLC-MS, Amycolatopsis sp. FJNU1011 can produce not only kigamicins, but also one new uncharacterized analogue of kigam icins.

  6. Pyrosequencing of mcrA and archaeal 16S rRNA genes reveals diversity and substrate preferences of methanogen communities in anaerobic digesters.

    Science.gov (United States)

    Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng; Lee, Patrick K H

    2015-01-01

    Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield.

  7. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements.

    Science.gov (United States)

    Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

    2006-10-01

    As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome

  8. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    Science.gov (United States)

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  9. [Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

    Science.gov (United States)

    Nazina, T N; Shumkova, E S; Sokolova, D Sh; Babich, T L; Zhurina, M V; Xue, Yan-Fen; Osipov, G A; Poltaraus, A B; Tourova, T P

    2015-01-01

    The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

  10. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia.

    Science.gov (United States)

    Xia, Li-Ping; Bian, Long-Yan; Xu, Min; Liu, Ying; Tang, Ai-Ling; Ye, Wen-Qin

    2015-01-01

    Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.

  11. Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

    Science.gov (United States)

    Ram, Jeffrey L; Karim, Aos S; Sendler, Edward D; Kato, Ikuko

    2011-06-01

    Understanding the identity and changes of organisms in the urogenital and other microbiomes of the human body may be key to discovering causes and new treatments of many ailments, such as vaginosis. High-throughput sequencing technologies have recently enabled discovery of the great diversity of the human microbiome. The cost per base of many of these sequencing platforms remains high (thousands of dollars per sample); however, the Illumina Genome Analyzer (IGA) is estimated to have a cost per base less than one-fifth of its nearest competitor. The main disadvantage of the IGA for sequencing PCR-amplified 16S rRNA genes is that the maximum read-length of the IGA is only 100 bases; whereas, at least 300 bases are needed to obtain phylogenetically informative data down to the genus and species level. In this paper we describe and conduct a pilot test of a multiplex sequencing strategy suitable for achieving total reads of > 300 bases per extracted DNA molecule on the IGA. Results show that all proposed primers produce products of the expected size and that correct sequences can be obtained, with all proposed forward primers. Various bioinformatic optimization of the Illumina Bustard analysis pipeline proved necessary to extract the correct sequence from IGA image data, and these modifications of the data files indicate that further optimization of the analysis pipeline may improve the quality rankings of the data and enable more sequence to be correctly analyzed. The successful application of this method could result in an unprecedentedly deep description (800,000 taxonomic identifications per sample) of the urogenital and other microbiomes in a large number of samples at a reasonable cost per sample.

  12. Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

    Science.gov (United States)

    Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

    2014-10-15

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

    KAUST Repository

    Prest, Emmanuelle I E C

    2014-10-01

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.

  14. Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene.

    Science.gov (United States)

    Motoshima, Maiko; Yanagihara, Katsunori; Morinaga, Yoshitomo; Matsuda, Junichi; Hasegawa, Hiroo; Kohno, Shigeru; Kamihira, Shimeru

    2012-11-01

    Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

  15. Assessment of rpoB and 16S rRNA Genes as Targets for PCR-Based Identification of Pasteurella pneumotropica

    OpenAIRE

    Dole, Vandana S.; Banu, Laila A; Fister, Richard D; Nicklas, Werner; Henderson, Kenneth S.

    2010-01-01

    Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica fr...

  16. Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Youjin Hu

    2013-01-01

    Full Text Available Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells’ proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.

  17. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns.

    Science.gov (United States)

    Atherly, Todd; Ziemer, Cherie J

    2014-04-01

    One-hundred-and-three isolates of Bacteroides ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade.

  18. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    Science.gov (United States)

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  19. Combined analyses of the ITS loci and the corresponding 16S rRNA genes reveal high micro- and macrodiversity of SAR11 populations in the Red Sea.

    KAUST Repository

    Ngugi, David

    2012-11-20

    Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24 °C throughout the year, and a remarkable uniform temperature (~22 °C) and salinity (~41 psu) from the mixed layer (~200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea\\'s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.

  20. Combined analyses of the ITS loci and the corresponding 16S rRNA genes reveal high micro- and macrodiversity of SAR11 populations in the Red Sea.

    Directory of Open Access Journals (Sweden)

    David Kamanda Ngugi

    Full Text Available Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24 °C throughout the year, and a remarkable uniform temperature (~22 °C and salinity (~41 psu from the mixed layer (~200 m to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS region of SAR11 in different depths of the Red Sea's water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen on the population dynamics of this ubiquitous marine bacterium.

  1. Systematic use of universal 16S rRNA gene polymerase chain reaction (PCR) and sequencing for processing pleural effusions improves conventional culture techniques.

    Science.gov (United States)

    Insa, Rosario; Marín, Mercedes; Martín, Adoración; Martín-Rabadán, Pablo; Alcalá, Luís; Cercenado, Emilia; Calatayud, Laura; Liñares, Josefina; Bouza, Emilio

    2012-03-01

    Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.

  2. 16S rDNA Sequence Analysis of Representative Strains of Two Rhizobial New Groups Isolated from Astragalus spp.%两个黄芪根瘤菌新类群代表菌株的16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    辛玉华; 陈文新

    2002-01-01

    对黄芪根瘤菌两个新类群中心菌株CA8561和JL84进行了16S rDNA全序列测定,并进行了系统发育学分析.结果表明,CA8561位于Rhizobium分支中,JL84位于Sinorhizobium分支中.

  3. Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes.

    Science.gov (United States)

    Li, Meng; Cao, Huiluo; Hong, Yi-Guo; Gu, Ji-Dong

    2011-01-01

    The community and population dynamics of anammox bacteria in summer (wet) and winter (dry) seasons in estuarial mudflat sediment of the Mai Po Nature Reserve were investigated by 16S rRNA and hydrazine oxidoreductase (hzo) genes. 16S rRNA phylogenetic diversity showed that sequences related to 'Kuenenia' anammox bacteria were presented in summer but not winter while 'Scalindua' anammox bacteria occurred in both seasons and could be divided into six different clusters. Compared to the 16S rRNA genes, the hzo genes revealed a relatively uniform seasonal diversity, with sequences relating to 'Scalindua', 'Anammoxoglobus', and planctomycete KSU-1 found in both seasons. The seasonal specific bacterial groups and diversity based on the 16S rRNA and hzo genes indicated strong seasonal community structures in estuary sediment of this site. Furthermore, the higher abundance of hzo genes in summer than winter indicates clear seasonal population dynamics. Combining the physicochemical characteristics of estuary sediment in the two seasons and their correlations with anammox bacteria community structure, we proposed the strong seasonal dynamics in estuary sediment of Mai Po to be due to the anthropogenic and terrestrial inputs, especially in summer, which brings in freshwater anammox bacteria, such as 'Kuenenia', interacting with the coastal marine anammox bacteria 'Scalindua'.

  4. Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

    Science.gov (United States)

    Huys, Geert; Vanhoutte, Tom; Joossens, Marie; Mahious, Amal S; De Brandt, Evie; Vermeire, Severine; Swings, Jean

    2008-06-01

    The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.

  5. Species identification of meat products based on mtDNA 16S rRNA gene sequencing%应用mtDNA16S rRNA基因测序鉴定肉产品种属

    Institute of Scientific and Technical Information of China (English)

    艾斯卡尔·买买提; 马合木提·哈力克; 日沙来提·吐尔地; 古丽茹合萨·艾海提

    2011-01-01

    Objective Applied mtDNA 16S rRNA gene sequencing method performed species identification of meat products. Methods 15 unknown animal muscle samples were given for inspection by related authorities, pork, beef and lamb were bought and used as a control. Extract DNA by conventional phenol-chloroform method, amplified mtDNA 16S rRNA gene by using the general primer and the specific primers respectively, products were examined by 1. 5% agarose gel electrophoresis. Amplified sequences were sequenced by Shanghai Bio-Engineering Company and then compared the identities through sequencing and BLAST aligning. Results All PCR products from 15 received samples amplified with general primer were showed positive band; neither one of them had positive band while used the sheep specific primer; while used specific primer of pig and bovine 14 and 1 of testing samples had amplified positive band respectively. The identity of them with pork and beef were 96% ~ 100% respectively while sequenced and BLAST aligned. Conclusion Among the 15 unknown meat products 14were pork and one was beef.%目的 应用mtDNA 16S rRNA基因测序方法进行肉产品种属鉴定.方法 15份有关部门送检的未知动物肌肉样本,3份市购猪、牛和羊的肌肉为对照样本.常规酚-氯仿法提取模板DNA,分别用通用引物和特异性引物扩增mtDNA的16S rRNA基因片段,产物用1.5%琼脂糖电泳检测,扩增的基因片段由上海生物工程公司进行序列测定及基因库同源性匹配查询.结果 经通用引物扩增15份未知样本,均检测出阳性条带;用羊特异性引物扩增,无未知样本检出阳性条带;用猪及牛特异性引物扩增,分别有14份和1份样本检出阳性条带,经测序及同源性查询,分别与猪和牛的同源性为96%~100%.结论 送检未知肉产品样本中14份是猪肉,1份是牛肉.

  6. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    Science.gov (United States)

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  7. [Detection of bacterial signal of 16S rRNA gene in prostate tissues obtained by perineal approach from patient with chronic pelvic pain syndrome].

    Science.gov (United States)

    Zhu, Qing-Feng; Xie, Hui; Weng, Zhi-Liang; Yang, Yi-Rong; Chen, Bi-Cheng

    2009-03-31

    To explore the role of bacteria in etiology of chronic pelvic pain syndrome (CPPS), i.e., chronic prostatitis and the correlation between presence of bacterial signal of 16S ribosomal RNA (16S rRNA) gene and the response to antibiotics. Samples of prostatic and subcutaneous tissues were obtained by biopsy via perineal approach from 112 CPPS patients, aged 20 - 48. Polymerase chain reaction was conducted to detect the 16S rRNA gene of bacteria. The patients were treated with gatifloxacin 0.4 g once a day for 4 weeks and then 4 weeks later the effects of treatment were assessed by the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). PCR was completed in 94 of the 112 patients, and 18 were excluded because their subcutaneous biopsies were positive for 16S rRNA, showing the possible contamination of their prostatic tissues. The total positive rate of bacterial 16S rRNA gene was 63.8% (60/94). The positive rate of bacterial 16S rRNA gene in the patients with IIIa CPPS and IIIb CPPS were 68.3% and 60.3% respectively. The total gatifloxacin effective rate of positive bacterial signal group after the was 55.0%, significantly higher than that of the negative bacterial signal group (14.7%, P 6S rRNA positive IIIa CPPS patients was 75%, significantly higher than that of the 6S rRNA negative IIIa CPPS patients (23.1%, P 6S rRNA positive IIIb CPPS patients was 37.5%, significantly higher than that of the 6S rRNA negative IIIb CPPS patients (9.5%, P < 0.05). Bacterial infection is related to CPPS in part of the patients. Bacterial signal detection helps predict the effect of antimicrobial therapy.

  8. First report of neonatal bacteremia caused by "Haemophilus quentini" diagnosed by 16S rRNA gene sequencing, Italy.

    Science.gov (United States)

    Giufrè, Maria; Cardines, Rita; Degl'Innocenti, Roberto; Cerquetti, Marina

    2015-10-01

    We report the first case of neonatal bacteremia caused by a "Haemophilus quentini" isolate in Italy. The isolate was differentiated from H. influenzae by 16S rRNA sequencing and was characterized by comparison with the wild-type "H. quentini" CCUG 36167. Both isolates carried substitutions in penicillin-binding protein 3 but were susceptible to aminopenicillins.

  9. True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis

    DEFF Research Database (Denmark)

    Rudkjøbing, Vibeke Børsholt; Thomsen, Trine R; Alhede, Morten

    2011-01-01

    Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed. In c...

  10. Identification of a novel, invasive, not-yet-cultivated Treponema sp in the large intestine of pigs by PCR amplification of the 16S rRNA gene

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre;

    2006-01-01

    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii(T) (90.1%). The spirochete, here...

  11. Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera).

    Science.gov (United States)

    Chen, Rui; Wang, Zhe; Chen, Jing; Qiao, Ge-Xia

    2015-08-25

    It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research.

  12. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.;

    1997-01-01

    by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can...

  13. 16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting

    OpenAIRE

    Yendi Navarro-Noya; César Hernández-Rodríguez; Zenteno, Juan C.; Beatriz Buentello-Volante; Cancino-Díaz, Mario E.; Janet Jan-Roblero; Juan C. Cancino-Díaz

    2012-01-01

    Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases.

  14. Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

    Institute of Scientific and Technical Information of China (English)

    L(U) Fang; WANG Guangce

    2011-01-01

    Using shotgun sequencing data,the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao,China).There are two group Ⅰ introns in the 16S rRNA gene,which is structurally similar to that of Caulerpa sertularioides (Bryopsidales,Chlorophyta).The chloroplast-encoded tufA gene sequence is 1230 bp long,very AT-rich (61.5%),and is similar to previously published 16S rRNA sequences of bryopsidinean algae.Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae,Halimedineae,and Ostreobidineae are three distinct lineages.These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae.Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however,this clade lacked robust nodal support.Moreover,the phylogenetic tree inferred from rbcL GenBank sequences,combined with the geographical distributions of Bryopsis species,identified a strongly supportive clade for three differently distributed Asian Bryopsis species.The preliminary results suggesting that these organisms are of distinct regional endemism.

  15. Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

    NARCIS (Netherlands)

    Coolen, MJL; Post, E; Davis, CC; Forney, LJ

    2005-01-01

    To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the h

  16. Use of Partial 16S rRNA Gene Sequencing for Identification of Legionella pneumophila and Non-pneumophila Legionella spp.▿

    OpenAIRE

    Wilson, D. A.; Reischl, U.; Hall, G. S.; Procop, G. W.

    2006-01-01

    We examined 49 Legionella species, 26 L. pneumophila and 23 non-pneumophila Legionella spp., using partial 16S rRNA gene sequencing. This approach accurately identified all the L. pneumophila isolates, characterized all non-pneumophila Legionella isolates as such within this genus, and classified most (20/23; 87%) of the non-pneumophila Legionella isolates to the species level.

  17. [Archaeal diversity in permafrost deposits of Bunger Hills Oasis and King George Island (Antarctica) according to the 16S rRNA gene sequencing].

    Science.gov (United States)

    Karaevskaia, E S; Demchenko, L S; Demidov, N É; Rivkina, E M; Bulat, S A; Gilichinskiĭ, D A

    2014-01-01

    Archaeal communities of permafrost deposits of King George Island and Bunger Hills Oasis (Antarctica) differing in the content of biogenic methane were analyzed using clone libraries of two 16S rRNA gene regions. Phylotypes belonging to methanogenic archaea were identified in all horizons.

  18. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    Science.gov (United States)

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  19. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    Science.gov (United States)

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  20. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

    DEFF Research Database (Denmark)

    Wolff Sönksen, Ute; Christensen, Jens Jørgen; Nielsen, Lisbeth

    2010-01-01

    Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic charact...

  1. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    Science.gov (United States)

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  2. 一株抗重金属铜镉细菌的分离、鉴定及其16S rDNA的序列分析%Isolation, Identification and 16S rDNA Sequences Analysis of a Bacterial Resistant to Copper and Cadmium

    Institute of Scientific and Technical Information of China (English)

    潘园园; 陈雯莉; 黄巧云

    2005-01-01

    从湖北省大冶市矿区土壤中分离到一株高抗铜和镉的菌株,命名为NTG-01.该菌株可以单抗4.5mmol/L的铜和2mmol/L的镉,因此它是研究抗铜或镉机制的重要菌株.对分离到的NTG-01进行了形态学观察和生理生化鉴定以及16S rDNA序列分析.结果显示菌株NTG-01为细菌,革兰氏染色阴性,短杆状,鞭毛周生,菌体大小约为0.8μm×2.0μm,V-P实验阳性,甲基红实验阴性,利用葡萄糖产酸产气;通过对菌株NTG-01的16S rDNA序列进行测定和同源性比对,发现它与产气肠杆菌的16S rDNA有高达99%的同源性,结合形态和生理生化指标,将其鉴定为产气肠杆菌(Enterobacter aerogenes).通过测定NTG-01对9种重金属的MIC值,可知它对多种重金属具有普遍较高的抗性.

  3. First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis.

    Science.gov (United States)

    Chen, Lei; Zhang, Honghai; Liu, Guangshuai; Sha, Weilai

    2016-05-01

    The aim of this study was to investigate the bacterial community in the distal gut of dholes (Cuon alpinus) based on the analysis of bacterial 16S rRNA gene sequences. Fecal samples were collected from five healthy unrelated dholes captured from Qilian Mountain in Gansu province of China. The diversity of the fecal bacteria community was investigated by constructing a polymerase chain reaction (PCR)-amplified 16S rRNA gene clone library. Bacterial 16S rRNA gene was amplified by using universal bacterial primers 27F and 1492R. A total of 275 chimera-free near full length 16S rRNA gene sequences were collected, and 78 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified according to the 97 % sequence similarity. Forty-two OTUs (53.8 %) showed less than 98 % sequence similarity to 16S rRNA gene sequences reported previously. Phylogenetic analysis demonstrated that dhole bacterial community comprised five different phyla, with the majority of sequences being classified within the phylum Bacteroidetes (64.7 %), followed by Firmicutes (29.8 %), Fusobacteria (4.7 %),Proteobacteria (0.4 %), and Actinobacteria (0.4 %). The only order Bacteroidales in phylum Bacteroidetes was the most abundant bacterial group in the intestinal bacterial community of dholes. Firmicutes and Bacteroidetes were the two most diverse bacterial phyla with 46.2 and 44.9 % of OTUs contained, respectively. Bacteroidales and Clostridiales were the two most diverse bacterial orders that contained 44.9 and 39.7 % of OTUs, respectively.

  4. Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

    Science.gov (United States)

    Delforno, Tiago Palladino; Lacerda Júnior, Gileno Vieira; Noronha, Melline F; Sakamoto, Isabel K; Varesche, Maria Bernadete A; Oliveira, Valéria M

    2017-02-23

    The 16S rRNA gene amplicon and whole-genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide-spectrum profiles of microbial composition and metabolic diversity from a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one-carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

  5. 应用16S rDNA检测固定矫治患者牙周可疑病原菌变化研究%Changes of Periodontal Dubious Pathogens in Fixed Orthodontic Appliance Patients Using PCR on 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    戚琳; 张扬; 张丹; 王舰

    2006-01-01

    应用16S rDNA检测固定矫治患者龈下菌斑中牙周可疑病原,探讨戴用固定矫治器对牙周组织健康的影响.随机选择 36 例治疗时间超过 6 个月的固定矫治患者组成实验组,29 例未经正畸治疗者组成对照组.分别检验特定部位牙周临床指数并收集龈下菌斑样本.采用16S rDNA检测9种牙周可疑致病菌.实验组与对照组相比牙龈指数、牙周袋深度、探诊出血差异有明显统计学意义 (P<0.05);牙周可疑病原菌中牙龈卟啉菌、齿垢密螺旋体在实验组的检出率明显高于对照组(P<0.05).固定矫治器戴入可引起患者牙周可疑病原菌的明显变化.

  6. Phylogenetic Analysis and 16S rDNA of Yersinia enterocolitica Isolated from Yellow Catfish (Pelteobagrus fulvidraco)%黄颡鱼小肠结肠炎耶尔森氏菌16S rDNA及系统发育分析

    Institute of Scientific and Technical Information of China (English)

    苟小兰; 王利; 黄艳青

    2012-01-01

    从发病黄颡鱼(Pelteobagrus fulvidraco)心脏中分离到1株细菌GM 2402,进行了分离培养、形态学观察、生理生化试验与16S rDNA基因的PCR扩增及序列分析.结果表明,菌株GM 2402为革兰氏阴性杆菌,具动力性,V-P试验、水杨酸、过氧化氢酶、脲酶等阳性,不发酵鼠李糖、乳糖等,硫化氢、枸椽酸盐等阴性.16S rDNA序列长1 417 bp,GenBank的登录号是JX 101598,且与数据库中小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)的相似性高达99.6%,系统发育树显示与小肠结肠炎耶尔森氏菌聚为一个分支.综合菌株的生化特性及分子生物学分析结果,判定菌株GM2402为小肠结肠炎耶尔森氏菌.

  7. 16S rDNA analysis of Rhizobium fredii 15067 and Azotobacter chroococcum 10006%快生型大豆根瘤菌(Rhizobium fredii)15067与褐球固氮菌(Azotobacter chroococcum)10006的16S rDNA分析

    Institute of Scientific and Technical Information of China (English)

    李海英; 刘金玲; 张喜波; 康妮; 于海鹏; 李鹏

    2008-01-01

    分别对快生型大豆根瘤菌(Rhizobium fredii)15067和褐球固氮菌(Azotobacter chroococ-cum)10006两个菌株进行了16S rDNA的全序列测定,将此全序列与已知相关的16S rDNA进行了比较及聚类分析,得到系统发育树状图.在系统发育树中,根瘤菌15067隶属于中华根瘤菌属,与Sinorhizobium xinjiangense IAM 14142,SinoRhizobium fredii SjzZ 4构成一个分支,其中与S.fredii SjzZ4的相似性最高,达99.72%;固氮菌10006处于固氮菌属分支中,其中和Azotobacter chroococcum AM12A,Azotobacter chroococcum的相似性均高达99.93%.为确定两株菌株的分类地位、进一步发掘和利用菌株的优良性状提供了参考依据.

  8. Identification of the forensically important beetles Nicrophorus japonicus, Ptomascopus plagiatus and Silpha carinata (Coleoptera: Silphidae) based on 16S rRNA gene in China.

    Science.gov (United States)

    Tang, Z C; Guo, Y D; Zhang, X W; Shi, J; Yang, K T; Li, X L; Chen, Y Q; Cai, J F

    2012-09-01

    Sarcophagous beetles play an important role in estimating postmortem interval time (PMI) in the later stages decomposition of carcasses. However, the morphological similarity of beetles usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification is simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene from 15 beetles of Silphidae (Coleoptera), collected from 5 locations in 4 Chinese provinces, was evaluated. Phenogram analysis of the sequenced segments by the unweighted pairgroup method analysis (UPGMA) method showed that all specimens were properly assigned into four species with strong similarity, which indicated the possibility of separation congeneric species with the short 16S rRNA fragment. These results will be instrumental for implementation of the Chinese database of forensically relevant beetles.

  9. 中国大陆科学钻探(CCSD)地下岩心样品中的细菌群落分析%16S rDNA analysis of bacterial community in Donghai terrestrial deep subsurface: Microbiology research of the Chinese Continent Scientific Drilling

    Institute of Scientific and Technical Information of China (English)

    王远亮; 夏颖; 董海良; 东秀珠; 杨克迁; 黄力; 董志扬

    2005-01-01

    通过建立高效率的微生物未培养DNA提取技术和16 S rDNA分析技术,在中国大陆钻探深层岩芯中发现微生物存在的证据.从地下430.00米(S1)与1033.77米(S13)深度岩芯中分别提取到微生物DNA,进行16S rDNA PCR扩增,通过对S13样品DNA中克隆得到的54个16S rDNA的序列测定,初步构建了该深度地下微生物群落的系统进化树,发现了23种不同的微生物序列存在.通过DAPI染色证明在地下1033.77米的微生物数量约为1×104个/克岩石,采用实时定量PCR分析分析,发现了其中两种微生物(CCSD1305与CCSD1307)的数量为6.6×104个/g岩石与2.18×106个/g岩石.

  10. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota.

    Directory of Open Access Journals (Sweden)

    Lance B Price

    Full Text Available BACKGROUND: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007 and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. CONCLUSIONS/SIGNIFICANCE: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.

  11. Physiological characters and 16S rDNA sequence phylogenetic analysis of a pathogenic bacterium isolated from Spodoptera exigua Hünber%罹病甜菜夜蛾分离菌株的生理特征及16S rDNA系统发育分析

    Institute of Scientific and Technical Information of China (English)

    齐放军; 刘缨; 季志英

    2004-01-01

    从自然罹病死亡的甜菜夜蛾虫体中分离出甜菜夜蛾致病菌QL-1,对其30项生理特征分析结果表明,QL-1菌株与肠杆菌科(Enterobacteriaceae),沙雷氏菌属(Serratta)中的粘质沙雷氏菌(Serratia marcescens)的生理特征相符合.进一步用PCR扩增出QL-1的16S rDNA基因片段,测定其序列并进行系统发育分析,结果也表明,该菌株与Serratia marcescens处于同一进化树分支中,相似性达99.2%以上.

  12. Two genetic clusters in swine hemoplasmas revealed by analyses of the 16S rRNA and RNase P RNA genes.

    Science.gov (United States)

    Watanabe, Yusaku; Fujihara, Masatoshi; Obara, Hisato; Nagai, Kazuya; Harasawa, Ryô

    2011-12-01

    Only two hemoplasma species, Eperythrozoon parvum and Mycoplasma suis, have been recognized in pigs. Here we demonstrate the genetic variations among six hemoplasma strains detected from pigs, by analyzing the 16S rRNA and RNase P RNA (rnpB) genes, and propose a novel hemoplasma taxon that has not been described previously. Phylogenetic trees based on the nucleotide sequence of the 16S rRNA gene indicated that these six hemoplasmas were divided into two clusters representing M. suis and a novel taxon. We further examined the primary and secondary structures of the nucleotide sequences of the rnpB gene of the novel taxon, and found it distinct from that of M. suis. In conclusion, we unveiled a genetic cluster distinct from M. suis, suggesting a new swine hemoplasma species or E. parvum. Our findings also suggest that this novel cluster should be included in the genus Mycoplasma.

  13. Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

    Directory of Open Access Journals (Sweden)

    Bryan E. Dutton

    2002-12-01

    Full Text Available We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

  14. Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

    Directory of Open Access Journals (Sweden)

    Sarah M. Boomer

    2009-12-01

    Full Text Available We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.

  15. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

    Science.gov (United States)

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping

    2016-07-01

    The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae.

  16. Characterization of Enterococcus spp. from human and animal feces using 16S rRNA sequences, the esp gene, and PFGE for microbial source tracking in Korea.

    Science.gov (United States)

    Kim, Sei-Yoon; Lee, Jung Eun; Lee, Sunghee; Lee, Hee Tae; Hur, Ho-Gil; Ko, Gwangpyo

    2010-05-01

    Contamination from human and animal fecal waste is a primary cause of water pollution. Microbial source tracking (MST) may be a useful tool for high-quality environmental management and for assessing human health risks associated with water pollution. The goal of this study was to evaluate Enterococcus spp. as a target organism for MST. Thirty-four fecal samples were collected from five different sources (human, chicken, pig, cow, and goose) in South Korea. In total, 237 Enterococcus spp. were isolated from feces using membrane- Enterococcus indoxyl-beta-d-glucoside agar. The 16S rRNA gene and the whole genome were analyzed using nucleic acid sequencing and pulsed-field gel electrophoresis (PFGE), respectively. Both phylogenetic analysis and principal coordinate analysis using UniFrac were performed on the nucleic acid sequences of the 16S rRNA gene. According to P-tests from UniFrac, significant differences existed between Enterococcus spp. isolated from human feces and those from animal feces. In addition, we evaluated whether the esp gene of Enterococcus faecium could be a specific target for Enterococcus spp. isolated from human feces. Of 58 E. faecium isolates tested, only three were esp-positive. The specificity of the esp gene of E. faecium isolated from human feces was 100%, but the sensitivity was esp gene and 16S rRNA sequences, whereas PFGE provides limited information on the fecal sources of Enterococcus spp.

  17. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    Science.gov (United States)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  18. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites.

    Science.gov (United States)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-17

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg(-1) were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 10(3) to 2.28 × 10(6) and 4.17 × 10(2) to 1.99 × 10(5), respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  19. Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

    Science.gov (United States)

    Tong, Maomeng; Jacobs, Jonathan P; McHardy, Ian H; Braun, Jonathan

    2014-11-03

    Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

  20. 海南近海30株抗B16细胞活性放线菌的16S rDNA多样性分析%16S rDNA diversity analysis of 30 Streptomycetes isolates displaying significant cytotoxic activity against B16 cell from near-shore sediments of Hainan Island

    Institute of Scientific and Technical Information of China (English)

    闫莉萍; 洪葵; 胡申才; 刘丽华

    2005-01-01

    从海南近海,包括文昌红树林、海口红树林以及洋浦港等地采集样品,经苯酚、SDS、加热等预处理,稀释涂布麦芽汁-酵母膏琼脂(YE)、淀粉酪素琼脂(SC)、葡萄糖天冬氨酸琼脂(GA), 或者直接将样品稀释涂布加有重铬酸钾的高氏一号琼脂(Gause)和麦芽汁-酵母膏琼脂等进行平板分离.共获得354株放线菌,其中有76株具有不同程度的抗B16细胞毒活性.比较发现加热预处理法和重铬酸钾选择培养法对于广泛分离筛选抗肿瘤活性放线菌不失为一种快速、简便、行之有效的方法.YE、Gause培养基无论在分离到的放线菌总数,还是细胞毒活性菌株的比例上都显示了良好的效果.对30株具有较强抗B16细胞活性的链霉菌进行了扩增性16S rDNA限制性酶切片段多样性分析(16S ARDRA), 表明这30株链霉菌之间有较大的基因差异性.050642、060386和060524等3株菌序列分析进一步证明这3株菌属于链霉菌属,其中菌株050642与其亲缘关系最近的Streptomyces cattleya的相似性仅为95%,因此可能是一个新种.

  1. Analysis of DNA homology and 16S rDNA sequence of rhizobia, a new phenotypic subgroup, isolated from Xizang Autonomous Region of China%西藏根瘤菌新表观群的DNA同源性及16S rDNA全序列分析

    Institute of Scientific and Technical Information of China (English)

    王素英; 杨晓丽; 李海峰; 刘杰

    2006-01-01

    在前期数值分类工作的基础上,对7株与Rhizobium关系较密切的分离自西藏部分地区豆科植物Trigonella spp.和Astragalus spp.的根瘤菌所形成的独立表观群,通过DNA同源性测定及16S rDNA全序列分析进行了分类地位的进一步确定.结果表明:该独立表观群菌株的(G+C)mol%为59.5%~63.3%,群内菌株间DNA同源性在74.3%~92.3%之间,中心菌株XZ2-3与相关Rhizobium种之间的DNA同源性在0%~47.4%之间,是不同于Rhizobium内各种的新DNA同源群.另外,16S rDNA全序列分析结果也表明,中心菌株XZ2-3占居Rhizobium系统发育分支中的一个独立亚分支,其与临近R.leguminosarum USDA2370T和R.etli CFN42T之间的序列相似性分别为96.55%和96.62%.根据国际系统细菌学委员会提出的细菌种属分类标准,该独立表观群构成了一个不同于Rhizobium内各种的新种群.该研究结果丰富了现有根瘤菌分类系统,将为国际上现有Rhizobium的14个种中再增添一个新的分类单元.

  2. Phylogenetic Analysis of 16S rDNA Sequence and PCR - RFLP of Bacillus from Fumao - flavor Daqu%福矛高温大曲中芽孢杆菌16S rDNA-RFLP及系统发育分析

    Institute of Scientific and Technical Information of China (English)

    颜林春; 张守财; 马校卫; 汤二将; 黄祖新; 陈由强

    2012-01-01

    目的:从福建建瓯黄华山酿酒有限公司高温大曲中分离出89株芽孢杆菌,通过初步筛选鉴定并进行微生物多样性研究.方法:对其16S rDNA进行PCR - RFLP分析和系统发育研究.结果:初步筛选得到的18株芽孢杆菌被HhaⅠ和MspⅠ酶切聚类分为四大组.通过系统发育分析样品中有6株Bacillus subtilis,4株Bacillus cereus,2株Bacillus sonorensis,2株Bacillus licheniformis,以及Bacillus pumilus、Bacillus oleronius、Bacillus coagulans和Bacillus thuringiensis各1株.结论:研究显示该高温大曲中可培养芽孢杆菌具有微生物多样性.

  3. Analysis of 22 Strains of Nitrogen-fixing Filamentous Cyanobacteria with Heterocyst by Using PCR-RFLP of 16S rDNA%22种丝状异型胞固氮蓝细菌的16S rDNA的 PCR-RFLP 分析

    Institute of Scientific and Technical Information of China (English)

    陈坚; 吴忠兴; 郑伟文

    2002-01-01

    用1对引物CYA106F及781R(a)从23种蓝细菌的DNA中扩增出一条600 bp的16S rDNA-PCR片段.对该片段进行的限制性酶切长度多态性(RFLP)分析表明:可将其中21种自生或共生的丝状异型胞固氮蓝细菌的样品分为2个类群组,基本上对应于形态分类上的Anabaena及Nostoc属.但有1种被认为是Anabaena的蓝细菌以及作为对照的1种聚球藻(Synechococcus)蓝细菌样品被明显区分出来,并发现Anabaena属的蓝细菌也可同被子植物根乃拉草(Gunnera)形成共生固氮作用.

  4. 应用16S rDNA克隆文库法分析有机物料腐熟菌剂细菌组成%Bacteria Composition of Organic Matter-decomposing Inoculant Analyzed with 16S rDNA Clone Library

    Institute of Scientific and Technical Information of China (English)

    李国媛; 李俊; 姜昕; 李力; 沈德龙

    2007-01-01

    应用16S rDNA克隆文库法对有机物料腐熟菌剂A和B样品中的细菌组成进行分析研究.结果表明,样品A有14个OTU,主要是融合乳杆菌(Weissella confusa)、枯草芽孢杆菌(Bacillus subtilis)和短小芽孢杆菌(Bacillus pumilus),其比例分别占总克隆文库的28.6%、30.4%和23.2%;样品B有43个OTU,主要是布氏乳杆菌(Lactobacillus buchneri)、香肠乳杆菌(Lactobacillus farciminis)和耐酸乳杆菌(Lactobacillus acetotolerans),占总克隆文库的比例分别为18.03%、18.86%和13.12%;所得出的结果均与产品标注存在差异,样品A未提及细菌的种类,而样品B只标注短小芽孢杆菌.研究表明这一方法在微生物菌剂细菌组成分析及其质量检测中具有良好的应用前景.

  5. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    Energy Technology Data Exchange (ETDEWEB)

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  6. Expression of gyrB and 16S ribosomal RNA genes as indicators of growth and physiological activities of Legionella pneumophila.

    Science.gov (United States)

    Okuno, Toshihiro; Tani, Katsuji; Yamaguchi, Nobuyasu; Nasu, Masao

    2015-01-01

    To determine whether the DNA gyrase (gyrB) and 16S ribosomal RNA (16S rRNA) genes can be used as indicators of the biological activities of Legionella pneumophila, the expression levels were estimated. The ratio of mRNA/DNA in gyrB was 0.7 in mid log phase and decreased drastically after the log phase. For 16S rRNA, the ratio was highest in mid log phase (7.0×10(3)), and the value that was about 10% of that in the log phase was maintained for six days. The rRNA may be vital in the resting or active but nonculturable cells that are not growing but physiologically active. The expression levels of gyrB mRNA and 16S rRNA can be used as indicators of the growth activity and the physiological activity of L. pneumophila, respectively. Therefore, by measurement of these indicators, we can evaluate the activities of Legionella cells in various environments.

  7. [Characterizing Beijing's Airborne Bacterial Communities in PM2.5 and PM1 Samples During Haze Pollution Episodes Using 16S rRNA Gene Analysis Method].

    Science.gov (United States)

    Wang, Bu-ying; Lang, Ji-dong; Zhang, Li-na; Fang, Jian-huo; Cao, Chen; Hao, Ji-ming; Zhu, Ting; Tian, Geng; Jiang, Jing-kun

    2015-08-01

    During 8th-14th Jan., 2013, severe particulate matter (PM) pollution episodes happened in Beijing. These air pollution events lead to high risks for public health. In addition to various PM chemical compositions, biological components in the air may also impose threaten. Little is known about airborne microbial community in such severe air pollution conditions. PM2.5 and PM10 samples were collected during that 7-day pollution period. The 16S rRNA gene V3 amplification and the MiSeq sequencing were performed for analyzing these samples. It is found that there is no significant difference at phylum level for PM2.5 bacterial communities during that 7-day pollution period both at phylum and at genus level. At genus level, Arthrobacter and Frankia are the major airborne microbes presented in Beijing winter.samples. At genus level, there are 39 common genera (combined by first 50 genera bacterial of the two analysis) between the 16S rRNA gene analysis and those are found by Metagenomic analysis on the same PM samples. Frankia and Paracoccus are relatively more abundant in 16S rRNA gene data, while Kocuria and Geodermatophilus are relatively more abundant in Meta-data. PM10 bacterial communities are similar to those of PM2.5 with some noticeable differences, i.e., at phylum level, more Firmicutes and less Actinobacteria present in PM10 samples than in PM2.5 samples, while at genus level, more Clostridium presents in PM10 samples. The findings in Beijing were compared with three 16S rRNA gene studies in other countries. Although the sampling locations and times are different from each other, compositions of bacterial community are similar for those sampled at the ground atmosphere. Airborne microbial communities near the ground surface are different from those sampled in the upper troposphere.

  8. 16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa.

    Directory of Open Access Journals (Sweden)

    Hagen Frickmann

    Full Text Available The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation.Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture.Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture.Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required.

  9. Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

    OpenAIRE

    Tran, S D; Rudney, J. D.

    1996-01-01

    Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. S...

  10. RFLP Analysis of 16S rRNA Genes of Bacterial Community in Water Sample of a Petroleum Reservoir%油藏水样细菌群落16S rRNA基因的RFLP分析

    Institute of Scientific and Technical Information of China (English)

    陈平; 李辉; 牟伯中

    2005-01-01

    通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性.从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库.337 个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到 74 个操作分类单元 (OTUs) ,其中数量最多的 4 个OTUs共占克隆子总数的73.6%,另外 70 个OTUs的丰度均处于较低水平,有 57 个 OTUs 仅含有 1 个克隆子.结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性.

  11. Cloning and Sequencing of Phytoplasma 16S rRNA Gene From Infected Mulberry in China%桑黄化型萎缩病病原体16S rRNA基因的序列分析

    Institute of Scientific and Technical Information of China (English)

    夏志松; 難波成任

    2004-01-01

    用PCR法克隆了中国桑黄化型萎缩病病原植原体的16S rRNA基因,并进行了序列分析.结果表明:克隆的基因大小为1 372 bp,与日本桑萎缩病病原植原体16S rRNA的同源性高达99.85%,只存在个别碱基的突变.Blastj检索结果显示与中国桑黄化型萎缩病病原体16S rRNA的基因同源性高达99%的有来源于玉米、洋葱、土豆等50多种其它植物的植原体16S rRNA基因,表明植物萎缩病病原体16S rRNA基因具高度保守性.

  12. Application of 16S rRNA Gene Library Method in Bacterium Flora Analysis of Infected Wound%16S rRNA基因文库方法在感染标本菌群分析中的应用

    Institute of Scientific and Technical Information of China (English)

    常玉梅; 刘海燕

    2013-01-01

    目的 探讨16S rRNA基因文库方法对感染标本菌群的鉴定效果.方法 采集70例创伤患者的感染伤口脓液或渗出液标本,使用通用引物扩增标本中所有细菌的16S rRNA基因片段,构建16S rRNA基因文库,挑取阳性克隆测序,分析感染细菌的数量与种类.结果 从150个克隆子中检测7种不同酶切类型的克隆,鉴定出7类微生物,其中屎肠球菌种类最多,占88%,坚强肠球菌比例占2%,另5类序列与非可培养细菌类群的16S rRNA基因序列相似性较高,均占2%.结论 感染标本中屎肠球菌含量丰富,应用16S rRNA基因文库的方法可有效分离到感染标本中的非可培养菌.

  13. In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops.

    Science.gov (United States)

    Bredow, C; Azevedo, J L; Pamphile, J A; Mangolin, C A; Rhoden, S A

    2015-08-19

    Because of human population growth, increased food production and alternatives to conventional methods of biocontrol and development of plants such as the use of endophytic bacteria and fungi are required. One of the methods used to study microorganism diversity is sequencing of the 16S rRNA gene, which has several advantages, including universality, size, and availability of databases for comparison. The objective of this study was to analyze endophytic bacterial diversity in agricultural crops using published papers, sequence databases, and phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S rRNA gene was used to identify endophytic bacteria, in important agricultural crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes, located in different geographical regions (America, Europe, and Asia). The corresponding 16S rRNA gene sequences were selected from the NCBI database, aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The most common orders present in the analyzed cultures were Bacillales, Enterobacteriales, and Actinomycetales and the most frequently observed genera were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that only approximately 1.56% of the total sequences were not properly grouped, demonstrating reliability in the identification of microorganisms. This study identified the main genera found in endophytic bacterial cultures from plants, providing data for future studies on improving plant agriculture, biotechnology, endophytic bacterium prospecting, and to help understand relationships between endophytic bacteria and their interactions with plants.

  14. Bacterial characterization of Beijing drinking water by flow cytometry and MiSeq sequencing of the 16S rRNA gene.

    Science.gov (United States)

    Liu, Tingting; Kong, Weiwen; Chen, Nan; Zhu, Jing; Wang, Jingqi; He, Xiaoqing; Jin, Yi

    2016-02-01

    Flow cytometry (FCM) and 16S rRNA gene sequencing data are commonly used to monitor and characterize microbial differences in drinking water distribution systems. In this study, to assess microbial differences in drinking water distribution systems, 12 water samples from different sources water (groundwater, GW; surface water, SW) were analyzed by FCM, heterotrophic plate count (HPC), and 16S rRNA gene sequencing. FCM intact cell concentrations varied from 2.2 × 10(3) cells/mL to 1.6 × 10(4) cells/mL in the network. Characteristics of each water sample were also observed by FCM fluorescence fingerprint analysis. 16S rRNA gene sequencing showed that Proteobacteria (76.9-42.3%) or Cyanobacteria (42.0-3.1%) was most abundant among samples. Proteobacteria were abundant in samples containing chlorine, indicating resistance to disinfection. Interestingly, Mycobacterium, Corynebacterium, and Pseudomonas, were detected in drinking water distribution systems. There was no evidence that these microorganisms represented a health concern through water consumption by the general population. However, they provided a health risk for special crowd, such as the elderly or infants, patients with burns and immune-compromised people exposed by drinking. The combined use of FCM to detect total bacteria concentrations and sequencing to determine the relative abundance of pathogenic bacteria resulted in the quantitative evaluation of drinking water distribution systems. Knowledge regarding the concentration of opportunistic pathogenic bacteria will be particularly useful for epidemiological studies.

  15. Culturable actinobacteria from the marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by 16S rRNA gene-RFLP analysis.

    Science.gov (United States)

    Zhang, Haitao; Lee, Yoo Kyung; Zhang, Wei; Lee, Hong Kum

    2006-08-01

    A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification-restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.

  16. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

    Science.gov (United States)

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-08-01

    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms