scholarly journals FN-Identify: Novel Restriction Enzymes-Based Method for Bacterial Identification in Absence of Genome Sequencing

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Mohamed Awad ◽  
Osama Ouda ◽  
Ali El-Refy ◽  
Fawzy A. El-Feky ◽  
Kareem A. Mosa ◽  
...  
Keyword(s):  
16S Rrna ◽  
Genome Sequencing ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Rapid Progress ◽  
Restriction Maps ◽  
Sequencing Technology ◽  
Bacterial Populations

Sequencing and restriction analysis of genes like 16S rRNA and HSP60 are intensively used for molecular identification in the microbial communities. With aid of the rapid progress in bioinformatics, genome sequencing became the method of choice for bacterial identification. However, the genome sequencing technology is still out of reach in the developing countries. In this paper, we propose FN-Identify, a sequencing-free method for bacterial identification. FN-Identify exploits the gene sequences data available in GenBank and other databases and the two algorithms that we developed, CreateScheme and GeneIdentify, to create a restriction enzyme-based identification scheme. FN-Identify was tested using three different and diverse bacterial populations (members of Lactobacillus, Pseudomonas, and Mycobacterium groups) in an in silico analysis using restriction enzymes and sequences of 16S rRNA gene. The analysis of the restriction maps of the members of three groups using the fragment numbers information only or along with fragments sizes successfully identified all of the members of the three groups using a minimum of four and maximum of eight restriction enzymes. Our results demonstrate the utility and accuracy of FN-Identify method and its two algorithms as an alternative method that uses the standard microbiology laboratories techniques when the genome sequencing is not available.

scholarly journals Estimation of the Relative Abundance of DifferentBacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

1998 ◽  
Vol 64 (10) ◽  
pp. 3683-3689 ◽  
Author(s):  
Jacqueline Wood ◽  
Karen P. Scott ◽  
Gorazd Avguštin ◽  
C. James Newbold ◽  
Harry J. Flint
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Restriction Enzymes ◽  
Pcr Primers ◽  
Gut Contents ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Bacterial Populations

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.

AMPLIFIED RIBOSOMAL DNA RESTRICTION ANALYSIS (ARDRA) BAKTERI DENGAN POTENSI ANTIMIKROB YANG BERASOSIASI DENGAN SPONS Jaspis sp.

2010 ◽  
Vol 9 (1) ◽  
Author(s):  
Hermawaty Abubakar
Keyword(s):  
Genetic Diversity ◽  
Antimicrobial Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Dna ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Dna Restriction

<p><em>Sponges</em><em> are one of the components that compose coral reef which have a potential bioactive substance that has not been utilized. Sponges are generally able to survive in marine waters were nutrients are poor because of associations with other organisms, especially bacteria. This study aimed to isolate and characterize bacteria (endosymbiont and ectosimbion) that produce antimicrobial compounds, and analyze genetic diversity based on Amplified Ribosomal DNA Restriction Analysis (ARDRA). The results of isolation obtained 138 bacterial isolates, which are 70 endofit isolates and 68 surfaces isolates respectively. The results obtained, based on antimicrobial test, was 32 bacterial isolates (45.71%) of the total bacterial isolates that have endofit antimicrobial activity, whereas on the surface bacteria, 20 bacterial isolates (29.41%) of the total surface of the bacterial isolates also have antimicrobial activity. Genetic diversity was carried out on 30 isolates that has the best antimicrobial activity. Amply</em><em>fi</em><em>cation of 16S rRNA gene is done using specific primers, 63f and 1387r. The profile of 16S rRNA gene band shows a </em><em>high </em><em>diversity, which is generated after cutting with three restriction enzymes </em><em>i.e.</em><em> </em><em>RsaI</em><em>, HaeIII and HinfI. The three restriction enzymes have different cuts and properties. Construction of phylogenetic trees based on analysis of Amplified Ribosomal DNA restriction, grouped 30 isolates from the sponge Jaspis sp. which have a microbial activity on seven filotipe. This grouping is based on the similarities cuts of sites of each isolate after restriction by three different restriction enzymes.</em></p>

scholarly journals A novel subgroup 16SrVII-D phytoplasma identified in association with erigeron witches' broom

2015 ◽  
Vol 65 (Pt_8) ◽  
pp. 2761-2765 ◽  
Author(s):  
Daniela Flôres ◽  
Ana Paula de Oliveira Amaral Mello ◽  
Thays Benites Camargo Pereira ◽  
Jorge Alberto Marques Rezende ◽  
Ivan Paulo Bedendo
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Phytoplasma Strain ◽  
Total Dna ◽  
Virtual Rflp ◽  
The 16S Rrna Gene

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported ‘Candidatus Phytoplasma fraxini’-related strains ranged from 98 % to 99 % indicating the phytoplasma to be a strain affiliated with ‘Candidatus Phytoplasma fraxini’. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

scholarly journals The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

2020 ◽  
Vol 8 (1) ◽  
pp. 131 ◽  
Author(s):  
Leonardo Mancabelli ◽  
Christian Milani ◽  
Gabriele Andrea Lugli ◽  
Federico Fontana ◽  
Francesca Turroni ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
In Silico Analysis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Test Case ◽  
Bacterial Populations ◽  
Taxonomic Marker ◽  
The Impact

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

scholarly journals Genetic and Biochemical Diversity among Isolates ofPaenibacillus alvei Cultured from Australian Honeybee (Apis mellifera) Colonies

2000 ◽  
Vol 66 (3) ◽  
pp. 1098-1106 ◽  
Author(s):  
Steven P. Djordjevic ◽  
Wendy A. Forbes ◽  
Lisa A. Smith ◽  
Michael A. Hornitzky
Keyword(s):  
Apis Mellifera ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Cell ◽  
Restriction Profiles ◽  
Dna Profiles

ABSTRACT Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI,CfoI, AluI, FokI, andRsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in theHinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI,FokI, and HinfI differentiated P. alvei from the phylogenetically closely related speciesPaenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymesCfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity inP. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.

scholarly journals Non-Frankia Actinomycetes Isolated from Surface-Sterilized Roots of Casuarina equisetifolia Fix Nitrogen

2005 ◽  
Vol 71 (1) ◽  
pp. 460-466 ◽  
Author(s):  
Mar�a Vald�s ◽  
N�stor-Octavio P�rez ◽  
Paulina Estrada-de los Santos ◽  
Jes�s Caballero-Mellado ◽  
Juan Jos� Pe�a-Cabriales ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
Isotope Dilution Analysis ◽  
Acetylene Reduction Activity ◽  
Rrna Gene ◽  
Casuarina Equisetifolia ◽  
Dna Homology ◽  
16S Rrna Gene Sequences ◽  
Reduction Activity

ABSTRACT Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from 15N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.

Diets supplemented with chickpea or its main oligosaccharide component raffinose modify faecal microbial composition in healthy adults

2010 ◽  
Vol 1 (2) ◽  
pp. 197-207 ◽  
Author(s):  
W. Fernando ◽  
J. Hill ◽  
G. Zello ◽  
R. Tyler ◽  
W. Dahl ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Control Diet ◽  
Rflp Analysis ◽  
Healthy Adults ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Clone Libraries ◽  
Bacterial Populations ◽  
Significant Difference

The effects of diets supplemented with either chickpea or its main oligosaccharide raffinose on the composition of the faecal microbial community were examined in 12 healthy adults (18-65 years) in a randomised crossover intervention study. Subjects consumed their usual diet supplemented with soups and desserts that were unfortified, or fortified with either 200 g/d of canned chickpeas or 5 g/d of raffinose for 3 week periods. Changes in faecal bacterial populations of subjects were examined using 16S rRNA-based terminal restriction fragment length polymorphisms (T-RFLP) and clone libraries generated from the diet pools. Classification of the clone libraries and T-RFLP analysis revealed that Faecalibacterium prausnitzii, reported to be an efficient butyrate producer and a highly metabolically active bacterium in the human intestinal microbiota, was more abundant in the raffinose diet and the chickpea diet compared to the control diet. However, no significant difference was observed in the faecal total short chain fatty acid concentration or in the levels of the components (butyrate, acetate and propionate) with the chickpea diet or the raffinose diet compared to the control diet. Bifidobacterium species were detected by T-RFLP in all three diet groups and quantitative real-time PCR (qPCR) analysis showed a marginal increase in 16S rRNA gene copies of Bifidobacterium with the raffinose diet compared to control (P>0.05). The number of individuals showing TRFs for the Clostridium histolyticum - Clostridum lituseburense groups, which include pathogenic bacteria species and putrefactive bacteria, were lower in the chickpea diet compared to the other two treatments. Diet appeared to affect colonisation by a high ammonia-producing bacterial isolate which was detected in 83%, 92% and 42% of individuals in the control, raffinose and chickpea groups, respectively. Our results indicate that chickpea and raffinose have the potential to modulate the intestinal microbial composition to promote intestinal health in humans.

scholarly journals Bradyrhizobium ganzhouense sp. nov., an effective symbiotic bacterium isolated from Acacia melanoxylon R. Br. nodules

2014 ◽  
Vol 64 (Pt_6) ◽  
pp. 1900-1905 ◽  
Author(s):  
Jun Kun Lu ◽  
Ya Jing Dou ◽  
Ya Jie Zhu ◽  
Sheng Kun Wang ◽  
Xin Hua Sui ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
Phylogenetic Analyses ◽  
Jiangxi Province ◽  
Rrna Gene ◽  
Symbiotic Gene ◽  
Content Type ◽  
Acacia Melanoxylon ◽  
Link Type

Three slow-growing rhizobial strains, designated RITF806T, RITF807 and RITF211, isolated from root nodules of Acacia melanoxylon grown in Ganzhou city, Jiangxi Province, China, had been previously defined, based on amplified 16S rRNA gene restriction analysis, as a novel group within the genus Bradyrhizobium . To clarify their taxonomic position, these strains were further analysed and compared with reference strains of related bacteria using a polyphasic approach. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to ‘Bradyrhizobium rifense’ CTAW71, with a similarity value of 99.9 %. In phylogenetic analyses of the housekeeping and symbiotic gene sequences, the three strains formed a distinct lineage within the genus Bradyrhizobium , which was consistent with the results of DNA–DNA hybridization. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related species of the genus Bradyrhizobium , indicating that these three strains constituted a novel group distinct from any recognized species of the genus Bradyrhizobium . Based on the data obtained in this study, we conclude that our strains represent a novel species of the genus Bradyrhizobium , for which the name Bradyrhizobium ganzhouense sp. nov. is proposed, with RITF806T ( = CCBAU 101088T = JCM 19881T) as the type strain. The DNA G+C content of strain RITF806T is 64.6 mol% (T m).

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