Articoli di riviste: "16S rDNA"

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Fessehaie, A., S. H. De Boer e C. A. Lévesque. "Molecular characterization of DNA encoding 16S23S rRNA intergenic spacer regions and 16S rRNA of pectolyticErwiniaspecies". Canadian Journal of Microbiology 48, n. 5 (1 maggio 2002): 387–98. http://dx.doi.org/10.1139/w02-026.

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Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspeciessubspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.Key words: Erwinia spp., 16S rDNA, intergenic spacer region, tRNA genes, phylogeny.

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Fukatsu, Takema, e Naruo Nikoh. "Two Intracellular Symbiotic Bacteria from the Mulberry Psyllid Anomoneura mori (Insecta, Homoptera)". Applied and Environmental Microbiology 64, n. 10 (1 ottobre 1998): 3599–606. http://dx.doi.org/10.1128/aem.64.10.3599-3606.1998.

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ABSTRACT We characterized the intracellular symbiotic bacteria of the mulberry psyllid Anomoneura mori by performing a molecular phylogenetic analysis combined with in situ hybridization. In its abdomen, the psyllid has a large, yellow, bilobed mycetome (or bacteriome) which consists of many round uninucleated mycetocytes (or bacteriocytes) enclosing syncytial tissue. The mycetocytes and syncytium harbor specific intracellular bacteria, the X-symbionts and Y-symbionts, respectively. Almost the entire length of the bacterial 16S ribosomal DNA (rDNA) was amplified and cloned from the whole DNA ofA. mori, and two clones, the A-type and B-type clones, were identified by restriction fragment length polymorphism analysis. In situ hybridization with specific oligonucleotide probes demonstrated that the A-type and B-type 16S rDNAs were derived from the X-symbionts and Y-symbionts, respectively. Molecular phylogenetic analyses of the 16S rDNA sequences showed that these symbionts belong to distinct lineages in the γ subdivision of the Proteobacteria. No 16S rDNA sequences in the databases were closely related to the 16S rDNA sequences of the X- and Y-symbionts. However, the sequences that were relatively closely related to them were the sequences of endosymbionts of other insects. The nucleotide compositions of the 16S rDNAs of the X- and Y-symbionts were highly AT biased, and the sequence of the X-symbiont was the most AT-rich bacterial 16S rDNA sequence reported so far.

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Wang, Haiyin, Pengcheng Du, Juan Li, Yuanyuan Zhang, Wen Zhang, Na Han, Patrick C. Y. Woo e Chen Chen. "Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples". Journal of Medical Microbiology 63, n. 3 (1 marzo 2014): 433–40. http://dx.doi.org/10.1099/jmm.0.060616-0.

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Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1–1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

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Riley, Donald E., Richard E. Berger, David C. Miner e John N. Krieger. "Diverse and Related 16S rRNA-Encoding DNA Sequences in Prostate Tissues of Men with Chronic Prostatitis". Journal of Clinical Microbiology 36, n. 6 (1998): 1646–52. http://dx.doi.org/10.1128/jcm.36.6.1646-1652.1998.

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Treatment of chronic prostatitis/chronic pelvic pain syndrome is often empirical because clinical culture methods fail to detect prostate-associated pathogens in >90% of patients. Previously, we tested a variety of specific-microorganism PCRs and began a DNA sequence study after we found that 77% of prostatitis patients were PCR positive for prokaryotic rRNA-encoding DNA sequences (rDNAs) despite negative cultures using optimal techniques. In the present study, 36 rDNA clones from 23 rDNA-positive patients were sequenced. This study represents more than twice the total rDNA sequence and more than twice the number of patients in the previous study. The increased number of patients and clones sequenced allowed enhanced phylogenetic analyses and refinements in our view of rDNA species inhabiting the prostate. A continuum of related rDNAs that might be arbitrarily described as two major groups of rDNAs and several minor groups was found. Sequences termed Pros A, identified in 8 (35%) of 23 rDNA-positive patients, grouped withAeromonas spp. in phylogenetic studies. Sequences termed Pros B, identified in 17 (74%) of 23 rDNA-positive patients, were distinct from previously reported sequences, although all were >90% similar to known gram-negative bacteria. Of the nine patients for whom multiple rDNAs were sequenced, six had biopsy specimens containing rDNAs from more than one species. Four (17%) patients had rDNAs different from those of the Pros A and Pros B groups. Of these four, one patient had rDNA similar to that ofFlavobacterium spp., another had rDNA similar to that of Pseudomonas testosteroni, and two patients had rDNAs <70% similar to known rDNAs. These findings suggest that the prostate can harbor bacteria undetectable by traditional approaches. Most of these diverse sequences are not reported in environments outside the prostate. The sequence similarities suggest adaptation of limited groups of bacteria to the microenvironment of the prostate. Further studies may elucidate the relationship of prostate-associated bacteria to chronic prostatitis/chronic pelvic pain syndrome.

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Li, Yuanping, Yanrong Chen, Yaoning Chen, Yanxin Wu, Chun Zhang, Zhen Peng, Yihuan Liu, Sha Wang, Ran Xu e Ziping Zeng. "Effects of Physico-Chemical Parameters on Actinomycetes Communities during Composting of Agricultural Waste". Sustainability 11, n. 8 (13 aprile 2019): 2229. http://dx.doi.org/10.3390/su11082229.

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The objective of this study was to investigate the influence of physico-chemical parameters on Actinomycetes communities and to prioritize those parameters that contributed to Actinomycetes community composition during the composting of agricultural waste. Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR-DGGE) and redundancy analysis (RDA) were used to determine the relationships between those parameters and Actinomycetes community composition. Quantitative PCR (qPCR) and regression analysis were used to monitor the 16S rDNA copy numbers of Actinomycetes and to analyse the correlations between physico-chemical parameters and Actinomyces 16S rDNA gene abundance, respectively. The RDA results showed that moisture content, water soluble carbon (WSC) and pH (p < 0.05) made the main contributions to the temporal variations of Actinomycetes community composition. The output of the regression analysis indicated that moisture content (R2 = 0.407, p < 0.01) showed a negative linear relationship with the Actinomyces 16S rDNA gene abundance.

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Dahllöf, Ingela, Harriet Baillie e Staffan Kjelleberg. "rpoB-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity". Applied and Environmental Microbiology 66, n. 8 (1 agosto 2000): 3376–80. http://dx.doi.org/10.1128/aem.66.8.3376-3380.2000.

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ABSTRACT Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.

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BAO, Qiongli, Long-Jun DING, Yizong HUANG e Keqing XIAO. "Effect of rice straw and/or nitrogen fertiliser inputs on methanogenic archaeal and denitrifying communities in a typical rice paddy soil". Earth and Environmental Science Transactions of the Royal Society of Edinburgh 109, n. 3-4 (settembre 2018): 375–86. http://dx.doi.org/10.1017/s1755691018000580.

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ABSTRACTTo understand better the microbial functional populations which are involved in methanogenesis and denitrification in paddy soils with rice straw (RS) and/or nitrogen fertiliser (potassium nitrate, N) application, the dynamics of methanogens and the denitrifying community were monitored simultaneously during the incubation period. The results show that the community structure of methanogens remained relatively stable among treatments based on 16S rDNA analysis, but fluctuated based on 16S rRNA. The Methanocellaceae and Methanosarcinaceae dominated all treatments at 16S rDNA and 16S rRNA level, respectively. RS+N increased the relative abundance of Methanosaetaceae at the 16S rRNA level, while there was an increasing trend in that Methanomicrobiaceae following RS addition at the 16S rDNA level. RS and/or N did not significantly change the diversity of methanogens targeting both 16S rDNA and 16S rRNA. RS and RS+N increased copy numbers of methanogens targeting both 16S rDNA and 16S rRNA analyses. The community structure and abundance of nirS and nosZ-containing denitrifiers, and the diversity of nirS-containing denitrifiers was significantly altered only by the N treatment. These results indicate that the community structure, diversity and abundance of methanogens respond differently to RS addition at the 16S rDNA and 16S rRNA levels.

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Reischl, U., K. Feldmann, L. Naumann, B. J. M. Gaugler, B. Ninet, B. Hirschel e S. Emler. "16S rRNA Sequence Diversity in Mycobacterium celatum Strains Caused by Presence of Two Different Copies of 16S rRNA Gene". Journal of Clinical Microbiology 36, n. 6 (1998): 1761–64. http://dx.doi.org/10.1128/jcm.36.6.1761-1764.1998.

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Direct sequencing of the 16S rRNA gene (16S rDNA) ofMycobacterium celatum isolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.

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O'Donnell, Anthony G., e Heike E. Görres. "16S rDNA methods in soil microbiology". Current Opinion in Biotechnology 10, n. 3 (giugno 1999): 225–29. http://dx.doi.org/10.1016/s0958-1669(99)80039-1.

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Lamy, Brigitte, Fréderic Laurent e Angeli Kodjo. "Validation of a partialrpoBgene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources". Canadian Journal of Microbiology 56, n. 3 (marzo 2010): 217–28. http://dx.doi.org/10.1139/w10-006.

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A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum , nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

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Hashavya, S., I. Gross, A. Michael-Gayego, N. Simanovsky e R. Lamdan. "The efficacy of 16S ribosomal DNA sequencing in the diagnosis of bacteria from blood, bone and synovial fluid samples of children with musculoskeletal infections". Journal of Children's Orthopaedics 12, n. 2 (aprile 2018): 204–8. http://dx.doi.org/10.1302/1863-2548.12.170049.

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Background Musculoskeletal infections are among the most common bacterial infections in children leading to hospitalization, invasive procedures and prolonged antibiotic administration. Blood, synovial and sometimes tissue cultures are essential for the diagnosis and treatment of musculoskeletal infections; 16S ribosomal DNA (rDNA) sequencing is a novel diagnostic tool for the detection of bacteria. While the yield of 16S rDNA sequencing in synovial fluid was previously assessed, data regarding the efficacy of this method from blood samples or partially treated children with suspected musculoskeletal infections is lacking. In this study we assessed the yield of 16S rDNA sequencing in blood, bone and synovial samples of children with musculoskeletal infections. Methods Blood, synovial and bone samples were collected from children with suspected musculoskeletal infections and analyzed for the presence of 16S rDNA, the results were then compared with the benchmark microbial cultures. Results During the study period, 41 children (18 boys and 23 girls) with suspected acute musculoskeletal infection were enrolled. A positive blood culture was found in 6/31 cases (19.4%) with methicillin-susceptible Staphylococcus aureus being the most commonly isolated bacterium. No significant 16S rDNA detection in blood samples was recorded. Synovial fluid culture was positive in 6/28 samples (21%), Kingella kingae being the most common pathogen. When using the 16S rDNA sequencing method, the rate of positive results in synovial fluid was higher with bacterial detection in 12/23 (52%) samples. The 16S rDNA sequencing method was also able to identify pathogens in samples taken from partially treated children where cultures were negative with 16S rDNA detection in 5/5 samples. Conclusion Although 16S rDNA sequencing may increase the yield of bacterial detection in synovial samples of patients with musculoskeletal infections, there is no benefit from applying this method on blood samples. The 16S rDNA sequencing method may be particularly beneficial when antibiotic treatment was started prior to synovial fluid sampling. Level of Evidence Level-II diagnostic study

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Bauernfeind, Adolf, Ines Schneider, Renate Jungwirth e Carsten Roller. "Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis fromBurkholderia cepacia Genomovars I, III, and IV by PCR". Journal of Clinical Microbiology 37, n. 5 (1999): 1335–39. http://dx.doi.org/10.1128/jcm.37.5.1335-1339.1999.

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We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, andBurkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensiswere sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5′-AGC ACT CCC RCC TCT CAG-3′; for B. multivorans, 5′-AGC ACT CCC GAA TCT CTT-3′) and 23S rDNA primers (for B. vietnamiensis, 5′-TCC TAC CAT GCG TGC AA-3′) were paired with a general sense primer of 16S rDNAs (5′-AGR GTT YGA TYM TGG CTC AG-3′) or with a sense primer of 23S rDNA (5′-CCT TTG GGT CAT CCT GGA-3′). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.

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Asil, A., No author No author, S. Sanousi, M. Aljameel, H. Beir, A. Adam e M. Hamid. "Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan". Open Veterinary Journal 5, n. 2 (2014): 16. http://dx.doi.org/10.5455/ovj.2014.v4.i1.p16.

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This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated.

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Rodríguez-García, Raquel, María Ángeles Rodríguez-Esteban, Jonathan Fernández-Suárez, Ana Morilla, Enrique García-Carús, Mauricio Telenti, Carlos Morales, Guillermo Muñiz Albaiceta e Javier Fernández. "Evaluation of 16S rDNA Heart Tissue PCR as a Complement to Blood Cultures for the Routine Etiological Diagnosis of Infective Endocarditis". Diagnostics 11, n. 8 (30 luglio 2021): 1372. http://dx.doi.org/10.3390/diagnostics11081372.

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Identification of the causative pathogen is required to optimize the effective therapy in infective endocarditis (IE). The aim of this study was to assess a 16S rDNA PCR to identify bacteria from heart valve tissues and to evaluate its usefulness as a complement to blood and removed valves cultures. A total of 266 patients diagnosed with IE from January 2015 to December 2019 were evaluated. Results between 16S rDNA PCR from heart valve tissues were compared with microbiological cultures. Blood cultures were positive in 83.5% of patients diagnosed with IE, while 39.6% and 71.8% of the evaluated heart valve samples were positive by culture and 16S rDNA PCR, respectively. For 32 (12%) patients, 16S rDNA tissue PCR provided valuable information supporting the results of blood cultures in the case of bacteria characteristic from the skin microbiota. Additionally, a microorganism was identified by using 16S rDNA PCR in 36% of blood culture-negative cases. The present study reveals that molecular diagnosis using 16S rDNA tissue PCR provides complementary information for the diagnosis of IE, and it should be recommended in surgical endocarditis, especially when blood cultures are negative.

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Drancourt, Michel, Claude Bollet, Antoine Carlioz, Rolland Martelin, Jean-Pierre Gayral e Didier Raoult. "16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates". Journal of Clinical Microbiology 38, n. 10 (2000): 3623–30. http://dx.doi.org/10.1128/jcm.38.10.3623-3630.2000.

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Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter andPantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

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Reinoso, Elina, Silvana Dieser, Luis Calvinho, Cristina Bogni e Liliana Odierno. "Phenotyping and genotyping of streptococci in bovine milk in Argentinean dairy herds". Acta Veterinaria Hungarica 58, n. 3 (1 settembre 2010): 287–95. http://dx.doi.org/10.1556/avet.58.2010.3.2.

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Most veterinary and milk hygiene laboratories identify streptococci and enterococci based on serological and biochemical tests. The analysis of 16S rDNA was suggested to be used for more exact identification; however, its use has not been considered so far in monitoring studies. The objective of the present study was to compare a conventional phenotypic method with restriction fragment length polymorphism analysis of 16S rDNA (16S rDNA RFLP) for identification of streptococci isolated from composite milk samples collected in connection with intramammary infection (IMI) in six Argentinean dairy farms. Composite milk samples (n = 1223) from cows belonging to six herds were collected for bacteriological analysis. Twelve reference strains and fifty streptococci or streptococcuslike isolates were identified to species level by the API 20 Strep system, conventional biochemical tests and 16S rDNA RFLP in a blind assay. The remaining streptococci or streptococcus-like isolates (n = 40) were identified to the species level both by 16S rDNA RFLP and conventional biochemical tests. As indicated by Kappa values, agreement between the 16S rDNA RFLP and the conventional scheme for identification ofStreptococcus agalactiae, S. dysgalactiae, S. uberis, S. equinusandEnterococcus faecaliswas 0.91, 0.73, 0.92, 0.81 and 0.85, respectively. Together with the less frequently isolated streptococcal species, the conventional scheme correctly identified 77 out of 90 isolates (85.5%). Thus, the use of 16S rDNA RFLP is considered valuable for monitoring studies due to its affordable cost for standard laboratories.

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Björnsson, Lovisa, Philip Hugenholtz, Gene W. Tyson e Linda L. Blackall. "Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal c cThe EMBL accession numbers for the sequences reported in this paper are X84472 (strain SBR1029 16S rDNA), X84474 (strain SBR1031 16S rDNA), X84498 (strain SBR1064 16S rDNA), X84565 (strain SBR2022 16S rDNA), X84576 (strain SBR2037 16S rDNA) and X84607 (strain SBR2076 16S rDNA)." Microbiology 148, n. 8 (1 agosto 2002): 2309–18. http://dx.doi.org/10.1099/00221287-148-8-2309.

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Hinrikson, Hans Peter, Fabrizio Dutly e Martin Altwegg. "Homogeneity of 16S-23S Ribosomal Intergenic Spacer Regions of Tropheryma whippelii in Swiss Patients with Whipple’s Disease". Journal of Clinical Microbiology 37, n. 1 (1999): 152–56. http://dx.doi.org/10.1128/jcm.37.1.152-156.1999.

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The current genetic strategies used to identify Tropheryma whippelii, the putative agent of Whipple’s disease, are based on PCR-mediated amplification of a part of its 16S rRNA gene (16S rDNA). Because there is very little intraspecies variation in these molecules, they are not suitable as targets for epidemiologic investigations. However, the intergenic spacer region between the 16S and 23S rDNAs is usually much more variable and has repeatedly been used for epidemiologic purposes. We have therefore amplified the spacer region of T. whippelii directly from clinical specimens from nine independent Swiss patients with Whipple’s disease by PCR with primers complementary to the 3′ and 5′ ends of the 16S and 23S rDNAs, respectively. The amplicons were directly sequenced and the sequences were compared to the T. whippelii reference sequence in GenBank/EMBL (accession no. X99636). Complete sequence homogeneity was found between the samples from our nine patients; the spacer sequence was also identical to the reference sequence. However, the sequences corresponding to the 3′ and 5′ ends of the 16S and the 23S rDNAs ofT. whippelii, respectively, differed from the respective sequences in GenBank/EMBL. The same sequence found in our patients was then found in a sample from the German patient from which the published sequence had been derived. We conclude that the 16S-23S rDNA spacer region seems to be very conserved in T. whippelii and that the respective reference entry in public databases should be revised.

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Patel, Amani, Kathryn A. Harris e Felicity Fitzgerald. "What is broad-range 16S rDNA PCR?" Archives of disease in childhood - Education & practice edition 102, n. 5 (17 aprile 2017): 261–64. http://dx.doi.org/10.1136/archdischild-2016-312049.

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Gosalbes, M. J., J. J. Abellan, A. Durban, A. E. Perez-Cobas, A. Latorre e A. Moya. "Metagenomics of human microbiome: beyond 16s rDNA". Clinical Microbiology and Infection 18 (luglio 2012): 47–49. http://dx.doi.org/10.1111/j.1469-0691.2012.03865.x.

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Grady, Ruth, Michael Anderson, David Drucker e David Denning. "Partial 16S rDNA analysis of oral Treponema". Reviews in Medical Microbiology 8 (1997): S25. http://dx.doi.org/10.1097/00013542-199712001-00012.

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Prüß, Birgit M., Kevin P. Francis, Felix von Stetten e Siegfried Scherer. "Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the Bacillus cereusGroup". Journal of Bacteriology 181, n. 8 (15 aprile 1999): 2624–30. http://dx.doi.org/10.1128/jb.181.8.2624-2630.1999.

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ABSTRACT Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.

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Ruiz, A., M. Poblet, A. Mas e J. M. Guillamón. "Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer." International Journal of Systematic and Evolutionary Microbiology 50, n. 6 (1 novembre 2000): 1981–87. http://dx.doi.org/10.1099/00207713-50-6-1981.

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Delbès, C., M. Leclerc, E. Zumstein, J. J. Godon e R. Moletta. "A molecular method to study population and activity dynamics in anaerobic digestors". Water Science and Technology 43, n. 1 (1 gennaio 2001): 51–57. http://dx.doi.org/10.2166/wst.2001.0013.

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Abstract (sommario):

The applicability of a new molecular fingerprinting method (Single Strand Conformation Polymorphism) to study the microbial populations of anaerobic digestors was investigated. After extraction of total nucleic acids, the 16S rDNA and 16S rRNA molecules were amplified and the amplicons were separated by SSCP electrophoresis. Characteristic and complex peak patterns were obtained, where each peak could be correlated with the 16S rDNA sequence of one micro-organism. The rDNA peak patterns should consist of the most abundant sequences and thus would reflect the diversity of prominent species of different digestors. Ribosomal DNA patterns were compared to rRNA patterns and revealed the bacteria that were the most active metabolically. The SSCP method also revealed dynamic changes in the presence and activity of populations, following perturbations such as an acidic shock which caused an increase in activity of two species. After cloning the 16S rDNA, the species corresponding to the peaks of interest, such as the archaeal species, could be identified by screening the clones according to their SSCP patterns and sequencing the 16S rDNA.

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25

Grahn, Niclas, Mounira Hmani-Aifa, Karin Fransén, Peter Söderkvist e Hans-Jürg Monstein. "Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis". Journal of Medical Microbiology 54, n. 11 (1 novembre 2005): 1031–35. http://dx.doi.org/10.1099/jmm.0.46122-0.

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Abstract (sommario):

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes’ classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.

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26

Lange, Carla C., Maria A. V. P. Brito, José R. F. Brito, Edna F. Arcuri, Guilherme N. Souza, Marco A. Machado, Robert Domingues e Alessandra P. S. Salimena. "Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero Staphylococcus isoladas de mastite bovina". Pesquisa Veterinária Brasileira 31, n. 1 (gennaio 2011): 36–40. http://dx.doi.org/10.1590/s0100-736x2011000100006.

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Abstract (sommario):

O objetivo deste trabalho foi identificar espécies de Staphylococcus (n=100) isoladas de mastite em rebanhos bovinos do Estado de Minas Gerais. Para esta finalidade foram utilizadas reações de PCR empregando oligonucleotídeos iniciadores descritos anteriormente para amplificar genes específicos de S. aureus (femA), S. intermedius (rDNA 16S) e S. hyicus (rDNA 16S-23S) e o sequenciamento do rDNA 16S. De acordo com as reações de PCR, 83 isolados foram identificados como S. aureus, 13 isolados como S. intermedius, dois como S. hyicus e dois isolados não foram identificados. Foram submetidos ao sequenciamento do rDNA 16S seis isolados identificados como S. aureus e os 17 restantes. Os seis isolados identificados como S. aureus confirmaram essa identificação. Dos outros 17 isolados, 13 foram identificados como S. chromogenes e quatro como S. hyicus, com similaridade igual ou superior a 99%. Baseando-se nos resultados da reação de PCR do gene femA e do sequenciamento do rDNA 16S, foram identificados 83 S. aureus, 13 S. chromogenes e quatro S. hyicus. Neste estudo os oligonucleotídeos iniciadores empregados na reação de PCR para S. intermedius não foram específicos, pois amplificaram também S. chromogenes; e os empregados na reação de PCR para S. hyicus não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus. A identificação definitiva das duas últimas espécies somente foi possível pelo sequenciamento do rDNA 16S.

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27

Devloo-Delva, Floriaan, Roger Huerlimann, Gladys Chua, Jordan K. Matley, Michelle R. Heupel, Colin A. Simpfendorfer e Gregory E. Maes. "How does marker choice affect your diet analysis: comparing genetic markers and digestion levels for diet metabarcoding of tropical-reef piscivores". Marine and Freshwater Research 70, n. 1 (2019): 8. http://dx.doi.org/10.1071/mf17209.

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Abstract (sommario):

Tropical reefs are highly diverse ecosystems, and reliable biomonitoring, through diet metabarcoding, is needed to understand present and future trophic relationships in this changing habitat. Several studies have assessed the reliability and effectiveness of single molecular markers; however, a cross-marker validation has rarely been performed. This study identified crucial properties for 12S rDNA, 16S rDNA and COI metabarcoding in tropical-reef piscivores (Plectropomus spp.). In addition, three new versatile primer sets for 16S were designed in silico for metabarcoding of reef fish. Results showed that COI was overall better at recovering true diversity because of a well-supported database. Second, optimal 16S amplicon sizes ranged between 160 and 440 base pairs for full diversity recovery, with increased species detection for the 270-base pairs region. Finally, blocking of predator-specific COI sequences was not equally effective in all host species, potentially introducing bias when diet compositions are directly compared. In conclusion, this novel study showed that marker success for prey identification is highly dependent on the reference database, taxonomic scope, DNA quality, amplicon length and sequencing platform. Results suggest that COI, complemented with 16S, yields the best outcome for diet metabarcoding in reef piscivores. Findings in this paper are relevant to other piscivores and other metabarcoding applications.

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28

Subandiyah, Siti, Toru Iwanami, Shinji Tsuyumu e Hiroyuki Ieki. "Comparison of 16S rDNA and 16S/23S Intergenic Region Sequences Among Citrus Greening Organisms in Asia". Plant Disease 84, n. 1 (gennaio 2000): 15–18. http://dx.doi.org/10.1094/pdis.2000.84.1.15.

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Abstract (sommario):

Polymerase chain reaction was used to amplify and sequence the 16S ribosomal RNA gene (rDNA) and 16S/23S intergenic region of several isolates of citrus greening organism (GO) from Japan, the Philippines, Indonesia, and Thailand. The sequences of 16S rDNA were identical among all the isolates studied, very similar to the published sequences of Thai (99.4 to 100% identity), Nepalese (100% identity), and Indian (98.8% identity) strains, and less similar to an African strain (97.5% identity). The sequences of the intergenic region between 16S and 23S rDNA were also identical among the isolates examined as well as the reported Nepalese and Thai isolates. They were close to the sequences of reported strains of India and China (99.2%) and apart from those of the African strain (85.5%). These results suggested that some isolates of GO from Japan, the Philippines, Indonesia, Thailand, and Nepal constitute one strain, which is similar to Indian and Chinese strains and distinct from the African strain.

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29

Heuer, Holger, Kathrin Hartung, Gabriele Wieland, Ina Kramer e Kornelia Smalla. "Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of Community Fingerprints". Applied and Environmental Microbiology 65, n. 3 (1 marzo 1999): 1045–49. http://dx.doi.org/10.1128/aem.65.3.1045-1049.1999.

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Abstract (sommario):

ABSTRACT Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5′ exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

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30

Chatellier, Sonia, Nathalie Mugnier, Françoise Allard, Bertrand Bonnaud, Valérie Collin, Alex van Belkum, Jean-Baptiste Veyrieras e Stefan Emler. "Comparison of two approaches for the classification of 16S rRNA gene sequences". Journal of Medical Microbiology 63, n. 10 (1 ottobre 2014): 1311–15. http://dx.doi.org/10.1099/jmm.0.074377-0.

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Abstract (sommario):

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.

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31

Ruta, Christine, Arne Nygren, Vincent Rousset, Per Sundberg, Annie Tillier, Helena Wiklund e Fredrik Pleijel. "Phylogeny of Hesionidae (Aciculata, Polychaeta), assessed from morphology, 18S rDNA, 28S rDNA, 16S rDNA and COI". Zoologica Scripta 36, n. 1 (gennaio 2007): 99–107. http://dx.doi.org/10.1111/j.1463-6409.2006.00255.x.

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32

MOHAMMED-GEBA, KHALED, EMAN M. ABBAS, HAMDY O. AHMED, MOHAMMED A. SHALABI, EL SAYED A. E. HAMED, FATMA A. ABDEL RAZEK e TAHA SOLIMAN. "Comparing genetic markers’ efficiencies for discrimination between two commercially important holothuroids in the Mediterranean Sea, Holothuria polii and Holothuria sanctori". Zootaxa 5092, n. 5 (24 gennaio 2022): 559–75. http://dx.doi.org/10.11646/zootaxa.5092.5.4.

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Abstract (sommario):

Sea cucumber (bêche-de-mer, Echinodermata: Holothuroidea) is one of the top internationally traded seafood varieties. Besides its direct nutritional benefits, it is continuously used in the traditional medicine in different areas and cultures in the world. This world-wide interest triggered various issues related to stocks´ declining and risks of species extinction. For these reasons, the current study was designed to provide molecular tools for accurate discrimination between two sea cucumber species that prevail the Mediterranean of these echinoderms in Egypt, that are Holothuria polii and H. sanctori. The power of three gene markers, i.e., 16S rDNA, 28S rDNA, and Histone H3 in achieving accurate DNA-based identification, as well as elucidating clear phylogenetic and genetic diversity differences between those two species was assessed. Among the three genes, 16S rDNA showed the highest potentials as genetic and phylogenetic species discrimination marker. Both 28S rDNA and H3 exhibited the least number of holothuroid reference sequences in the GenBank database. For genetic diversity within each species population, 16S rDNA exhibited the best potentials, followed by H3. 28S rDNA showed no genetic polymorphism at all. Moreover, the collective data of both H3 and 16S rDNA suggested a possible role of asexual reproduction behavior in H. sanctori in the reduction of genetic diversity, as a possible response to overfishing. Hence, the current research can recommend the simultaneous application of both 16S rDNA and H3 as accurate markers for genetic discrimination among H. polii, H. sanctori and other different holothuroid species.

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Marcone, Carmine, Bernd Schneider e Erich Seemüller. "‘Candidatus Phytoplasma cynodontis’, the phytoplasma associated with Bermuda grass white leaf disease". International Journal of Systematic and Evolutionary Microbiology 54, n. 4 (1 luglio 2004): 1077–82. http://dx.doi.org/10.1099/ijs.0.02837-0.

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Abstract (sommario):

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

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34

Allen, Julie M., J. Gordon Burleigh, Jessica E. Light e David L. Reed. "Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice". PeerJ 4 (19 luglio 2016): e2187. http://dx.doi.org/10.7717/peerj.2187.

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Abstract (sommario):

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 ofGammaproteobacteriasequences 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 otherGammaproteobacteria. 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.

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35

Satokari, Reetta M., Elaine E. Vaughan, Antoon D. L. Akkermans, Maria Saarela e Willem M. de Vos. "Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis". Applied and Environmental Microbiology 67, n. 2 (1 febbraio 2001): 504–13. http://dx.doi.org/10.1128/aem.67.2.504-513.2001.

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Abstract (sommario):

ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

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36

Wood, Jacqueline, Karen P. Scott, Gorazd Avguštin, C. James Newbold e Harry J. Flint. "Estimation of the Relative Abundance of DifferentBacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences". Applied and Environmental Microbiology 64, n. 10 (1 ottobre 1998): 3683–89. http://dx.doi.org/10.1128/aem.64.10.3683-3689.1998.

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Abstract (sommario):

ABSTRACT We describe an approach for determining the genetic composition ofBacteroides and Prevotellapopulations 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 andPrevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution ofBacteroides and Prevotellasequences 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 Prevotellastrains, together accounted for between 20 and 86% of the total amplified Bacteroides andPrevotella rDNA in these samples. The most abundantBacteroides 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 abundantBacteroides and Prevotella groups in the rumen are underrepresented among cultured rumenPrevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

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37

Zoetendal, Erwin G., Antoon D. L. Akkermans e Willem M. De Vos. "Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria". Applied and Environmental Microbiology 64, n. 10 (1 ottobre 1998): 3854–59. http://dx.doi.org/10.1128/aem.64.10.3854-3859.1998.

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Abstract (sommario):

ABSTRACT The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from differentClostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.

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38

Li, Yong Feng, Yi Xuan Wang, Lu Wang e Zhan Qing Wang. "Sequence Length Variation of Internal Genic Space of 16S rDNA-23S rDNA in Bacterium with High Yield of Hydrogen Production". Advanced Materials Research 183-185 (gennaio 2011): 1413–16. http://dx.doi.org/10.4028/www.scientific.net/amr.183-185.1413.

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Abstract (sommario):

To develop the identification of species for fermentative biohydrogen-producing bacterium, scholars have found a method which is based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions. In the study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2000 bases of the 23S rDNA, were polymerasechain reaction (PCR) amplified. The PCR amplification of the genomic DNA of Leptonema ilk strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases.

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39

Ritchie, Nancy J., e David D. Myrold. "Phylogenetic placement of unculturedCeanothusmicrosymbionts using 16S rRNA gene sequences". Canadian Journal of Botany 77, n. 9 (18 dicembre 1999): 1208–13. http://dx.doi.org/10.1139/b99-080.

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Abstract (sommario):

Full-length 16S rDNA sequences were amplified directly from the nodules of Ceanothus americanus L. and Ceanothus thyrsiflorus Eschsch. using the polymerase chain reaction. Sequences were determined using an automated sequencer, compared against those in GenBank, and assembled into consensus sequences. The sequences were aligned with other full-length Frankia 16S rDNA sequences available from the data base. Phylogenetic trees were obtained using three different algorithms: neighbor joining, parsimony, and the maximum-likelihood method. All three methods showed that these Ceanothus L. microsymbionts were most closely related to the microsymbiont associated with Dryas drummondii Richardson ex Hook. Lvs. rather than Frankia isolated from the Elaeagnaceae.Key words: Frankia, Ceanothus, 16S rDNA.

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40

Vinay, Oraon, Prasad Bhupendra e Singh Kiran. "16S rDNA-RFLP analysis of phylogenetic tree of Rhizobium bacteria". Indian Journal of Applied Research 3, n. 12 (1 ottobre 2011): 474–76. http://dx.doi.org/10.15373/2249555x/dec2013/145.

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41

G. D.Sharma, G. D. Sharma, *. Dhritiman Chanda e D. K. Jha D.K. Jha. "16S rDNA Sequence based Phylogenetic Analysis of Some Bacterial Phytoplasma". International Journal of Scientific Research 3, n. 3 (1 giugno 2012): 302–4. http://dx.doi.org/10.15373/22778179/march2014/101.

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42

Robinson, K. G., H. M. Dionisi, G. Harms, A. C. Layton, I. R. Gregory e G. S. Sayler. "Molecular assessment of ammonia- and nitrite-oxidizing bacteria in full-scale activated sludge wastewater treatment plants". Water Science and Technology 48, n. 8 (1 novembre 2003): 119–26. http://dx.doi.org/10.2166/wst.2003.0460.

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Abstract (sommario):

Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 × 1013 copies/L and Nitrospira 16S rDNA values were 2-4 × 1010 copies/L. amoA gene concentrations averaged 1.73 × 109 copies/L (municipal) and 1.06 × 1010 copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.

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43

Tanner, Michael A., Brett M. Goebel, Michael A. Dojka e Norman R. Pace. "Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants". Applied and Environmental Microbiology 64, n. 8 (1 agosto 1998): 3110–13. http://dx.doi.org/10.1128/aem.64.8.3110-3113.1998.

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Abstract (sommario):

ABSTRACT Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella(formerly Zoogloea), Acinetobacter,Stenotrophomonas, Escherichia,Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.

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44

Texeira, D. C., J. Ayres, E. W. Kitajima, L. Danet, S. Jagoueix-Eveillard, C. Saillard e J. M. Bové. "First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, “Candidatus Liberibacter americanus”, with the Disease". Plant Disease 89, n. 1 (gennaio 2005): 107. http://dx.doi.org/10.1094/pd-89-0107a.

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Huanglongbing (HLB) (ex-greening) is one of the most serious diseases of citrus. The causal agent is a noncultured, sieve tube-restricted α-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia (2). The disease has never been reported from the American continent. However, Diaphorina citri, the Asian psyllid vector of HLB, is found in South, Central, and North America (Florida and Texas). Early in 2004, leaf and fruit symptoms resembling those of HLB were observed in several sweet orange orchards near the city of Araraquara, Sao Paulo State. Leaf mottling on small and large leaves was the major symptom. Shoots with affected leaves were yellowish. Fruits were small and lopsided, contained many aborted seeds, and appeared more severely affected than were plants infected with classic HLB. Forty-three symptomatic samples and twenty-five samples of symptomless sweet orange leaves from five farms were analyzed for the presence of the HLB-liberibacters using polymerase chain reaction (PCR) with two sets of HLB-specific primers for amplification of 16S rDNA (2,3) and ribosomal protein genes (1). None of the 43 symptomatic leaf samples gave a positive PCR amplification, while HLB-affected leaves from the Bordeaux HLB collection produced the characteristic amplicons with both sets of primers. The 43 symptomatic and the 25 symptomless leaf samples were then analyzed using PCR with universal primers for amplification of bacterial 16S rDNA (4). All symptomatic leaf samples, but none of the symptomless leaf samples, yielded the same 16S rDNA amplification product, indicating the presence of a bacterium in the symptomatic leaves. This was confirmed using the observation of a sieve tube restricted bacterium by electron microscopy. The 16S rDNA product was cloned, sequenced, and compared with those of “Ca. L. africanus” and “Ca. L. asiaticus”. While the 16S rDNAs of these two liberibacter species have 97.5% sequence identity, the 16S rDNA sequence of the new bacterium shared only 93.7% identity with that of “Ca. L. asiaticus” and 93.9% with that of “Ca. L. africanus”. The 16S rDNA sequence of the new bacterium had a secondary loop structure characteristic of the α subdivision of the proteobacteria and possessed all the oligonucleotide signatures characteristic of the liberibacters. For these reasons, the new bacterium is a liberibacter and is sufficiently different phylogenetically from known liberibacters to warrant a new species, “Candidatus Liberibacter americanus”. Specific PCR primers for amplification of the 16S rDNA of the new species have been developed. They were able to detect “Ca. L. americanus” in 214 symptomatic leaf samples from 47 citrus farms in 35 municipalities, while the “old” species, “Ca. L. asiaticus”, has been found only four times within the 47 farms. References: (1) A. Hocquellet et al. Mol. Cell. Probes, 13:373, 1999. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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Ramírez-Saad, Hugo, Jaap D. Janse e Antoon DL Akkermans. "Root nodules ofCeanothus caeruleuscontain both the N2-fixingFrankiaendophyte and a phylogetically related Nod-/Fix-actinomycete". Canadian Journal of Microbiology 44, n. 2 (1 febbraio 1998): 140–48. http://dx.doi.org/10.1139/w97-138.

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Attempts to isolate the N2-fixing endophyte of Ceanothus caeruleus (Rhamnaceae) root nodules, led to the isolation of nine actinomycetous strains. Owing to their inability to fix nitrogen (Fix-) and nodulate (Nod-), they could not be regarded as the effective endophyte. Characterization was done based on morphological and physiological features and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and sequencing of nearly full length 16S rDNA and partial nifH genes. Phylogenetic analysis based on 16S rDNA revealed that both the effective endophyte and the isolated actinomycetes belong to two different but well-defined lineages within the family Frankiaceae. One lineage is formed mainly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix-/Nod-isolates. Application of temperature gradient gel electrophoresis techniques to actinorhizal nodules allowed us to detect and identify 16S rDNA sequences from both the Fix+and the Fix-nodule inhabitants. Interestingly, these same two sequences were detected on Hippophae rhamnoides nodules obtained after inoculation with Ceanothus caeruleus nodule suspensions. The isolates were located in the outer layers of the nodule.Key words: Frankia, Ceanothus, 16S rDNA, nifH, temperature gradient gel electrophoresis (TGGE), Fix-/Nod-strains.

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46

Jomantiene, R. R., J. L. Maas, F. Takeda e R. E. Davis. "Molecular Identification and Classification of Strawberry Phylloid Fruit Phytoplasma in Group 16SrI, New Subgroup". Plant Disease 86, n. 8 (agosto 2002): 920. http://dx.doi.org/10.1094/pdis.2002.86.8.920c.

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Plants of commercial strawberry (Fragaria × ananassa Duch., cv. Camarosa) exhibiting extensive fruit phyllody (development of leafy structures from achenes) were observed in a winter greenhouse production facility in West Virginia. In July 2001, 95 dormant, cold-stored plants were purchased from a California strawberry nursery, potted and grown in this West Virginia facility. Five of the plants developed fruits with phylloid growths. These fruits were assessed for phytoplasma infection using nested polymerase chain reactions (PCRs) in which initial ribosomal (r) DNA amplification was primed by phytoplasma-universal primer pair P1/P7 (2), and rDNA reamplification was primed by primer pair R16F2n/R16R2 (1). Amplification of phytoplasma-characteristic 1.2-kbp 16S rDNA in the nested reactions primed by R16F2n/R16R2 confirmed that the symptomatic plants were infected by a phytoplasma, termed strawberry phylloid fruit (StrawbPhF) phytoplasma. No phytoplasma DNAs were amplified from healthy plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested with AluI, KpnI, HhaI, HaeIII, HpaII, MseI, RsaI, and Sau3A1 restriction endonucleases indicated that StrawbPhF phytoplasma belonged to group 16SrI (group I, aster yellows phytoplasma group) according to the phytoplasma classification system of Lee et al. (4). However, the collective patterns distinguished StrawbPhF from its closest known relative, clover phyllody (CPh) phytoplasma, and from all other phytoplasmas classified in group 16SrI. On the basis of the RFLP patterns of 16S rDNA, the StrawbPhF was classified in group 16SrI, new subgroup R. The StrawbPhF phytoplasma 1.2-kbp 16S rDNA PCR product was cloned in Escherichia coli using TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), sequenced, and the sequence deposited in GenBank under Accession No. AY102275. The StrawbPhF 16S rDNA sequence shared 99.9 and 99.8% similarity with the two sequence heterogeneous operons, rrnA and rrnB, respectively, of CPh phytoplasma, and shared 99.9% similarity with 16S rDNA of the unclassified cirsium yellows (CirY) phytoplasma (GenBank Accession No. AF200431) reported in Cirsium arvense L. in Lithuania (3). The restriction sites in 16S rDNA of StrawbPhF were identical to those in 16S rDNA of CPh rrnA and CirY. Three restriction sites (AluI, HaeIII, and MseI) and three base substitutions distinguished StrawbPhF 16S rDNA from rrnB of CPh phytoplasma. No evidence was obtained for the presence of a second (sequence heterogeneous) rRNA operon in StrawbPhF phytoplasma, as reported in CPh phytoplasma (4), which clearly distinguishes this phytoplasma from CPh phytoplasma. Future studies on StrawbPhF phytoplasma may provide important information on the evolution of phytoplasmas. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) R. Jomantiene et al. Phytopathology 90:S39, 2000. (4) I.-M. Lee et al. Int J. Syst. Bacteriol. 48:1153, 1998.

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Fu, Yue, Xiang-Liang Fang, Xin-Hua Wang, Mi Shen e Yun-Li Xiao. "Corynoneura Winnertz species from Hunan Province, Oriental China, delineated with morphological and 16S rDNA data (Diptera, Chironomidae)". ZooKeys 1082 (19 gennaio 2022): 87–102. http://dx.doi.org/10.3897/zookeys.1082.73019.

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The genus Corynoneura Winnertz, 1846 from Hunan Province in Oriental China is reviewed. Four new species, C. enormis Fu sp. nov., C. gibbera Fu sp. nov., C. incuria Fu sp. nov., and C. longshanensis Fu sp. nov. are described and illustrated based on adult males. Sequence data from the 16S rDNA gene were used to infer relationships between these species and complement morphological delineation. Sequences from the mitochondrial large ribosomal subunit (16S rDNA) from these species are uploaded to the National Center for Biotechnology Information (NCBI). Relationships were inferred using the Neighbor-Joining method based on 16S rDNA.

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48

BjÃ¯Â¿Â½rkroth, K. Johanna, Rolf Geisen, Ulrich Schillinger, Norbert Weiss, Paul De Vos, Wilhelm H. Holzapfel, Hannu J. Korkeala e Peter Vandamme. "Characterization of Leuconostoc gasicomitatum sp. nov., Associated with Spoiled Raw Tomato-Marinated Broiler Meat Strips Packaged under Modified-Atmosphere Conditions". Applied and Environmental Microbiology 66, n. 9 (1 settembre 2000): 3764–72. http://dx.doi.org/10.1128/aem.66.9.3764-3772.2000.

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ABSTRACT Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by aLeuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentativeLactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called “protein swell.” This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominantLeuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the nameLeuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.

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Vinuesa, Pablo, Jan L. W. Rademaker, Frans J. de Bruijn e Dietrich Werner. "Genotypic Characterization ofBradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing". Applied and Environmental Microbiology 64, n. 6 (1 giugno 1998): 2096–104. http://dx.doi.org/10.1128/aem.64.6.2096-2104.1998.

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ABSTRACT We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulatedGlycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus,Macroptilium atropurpureum, and Vigna unguiculata.

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Janz, V., J. Schoon, C. Morgenstern, B. Preininger, S. Reinke, G. Duda, A. Breitbach, C. F. Perka e S. Geissler. "Rapid detection of periprosthetic joint infection using a combination of 16s rDNA polymerase chain reaction and lateral flow immunoassay". Bone & Joint Research 7, n. 1 (gennaio 2018): 12–19. http://dx.doi.org/10.1302/2046-3758.71.bjr-2017-0103.r2.

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Objectives The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI). Methods The 16s rDNA test combines a polymerase chain reaction (PCR) for amplification of 16s rDNA with a lateral flow immunoassay in one fully automated system. The synovial fluid of 77 patients undergoing joint aspiration or primary or revision total hip or knee surgery was prospectively collected. The cohort was divided into a proof-of-principle cohort (n = 17) and a validation cohort (n = 60). Using the proof-of-principle cohort, an optimal cut-off for the discrimination between PJI and non-PJI samples was determined. PJI was defined as detection of the same bacterial species in a minimum of two microbiological samples, positive histology, and presence of a sinus tract or intra-articular pus. Results The 16s rDNA test proved to be very robust and was able to provide a result in 97% of all samples within 25 minutes. The 16s rDNA test was able to diagnose PJI with a sensitivity of 87.5% and 82%, and a specificity of 100% and 89%, in the proof-of-principle and validation cohorts, respectively. The microbiological culture of synovial fluid achieved a sensitivity of 80% and a specificity of 93% in the validation cohort. Conclusion The 16s rDNA test offers reliable intraoperative detection of all bacterial species within 25 minutes with a sensitivity and specificity comparable with those of conventional microbiological culture of synovial fluid for the detection of PJI. The 16s rDNA test performance is independent of possible blood contamination, culture time and bacterial species. Cite this article: V. Janz, J. Schoon, C. Morgenstern, B. Preininger, S. Reinke, G. Duda, A. Breitbach, C. F. Perka, S. Geissler. Rapid detection of periprosthetic joint infection using a combination of 16s rDNA polymerase chain reaction and lateral flow immunoassay: A Pilot Study. Bone Joint Res 2018;7:12–19. DOI: 10.1302/2046-3758.71.BJR-2017-0103.R2.

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