scholarly journals Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

2001 ◽  
Vol 67 (10) ◽  
pp. 4479-4487 ◽  
Author(s):  
L. Ranjard ◽  
F. Poly ◽  
J.-C. Lata ◽  
C. Mougel ◽  
J. Thioulouse ◽  
...  
Keyword(s):  
Length Distribution ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Internal Transcribed Spacers ◽  
Rrna Operon ◽  
Rrna Gene ◽  
Soil Community ◽  
Intergenic Spacer Region ◽  
5.8S Rrna Gene ◽  
Total Soil

ABSTRACT Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.

scholarly journals Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons

1999 ◽  
Vol 65 (9) ◽  
pp. 4264-4267 ◽  
Author(s):  
G. W. Tannock ◽  
A. Tilsala-Timisjarvi ◽  
S. Rodtong ◽  
J. Ng ◽  
K. Munro ◽  
...  
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Sequence Comparisons ◽  
Intergenic Spacer Region ◽  
23S Rrna Gene

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification ofLactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates ofLactobacillus casei and Lactobacillus rhamnosus.

scholarly journals Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities

1999 ◽  
Vol 65 (10) ◽  
pp. 4630-4636 ◽  
Author(s):  
Madeline M. Fisher ◽  
Eric W. Triplett
Keyword(s):  
Microbial Diversity ◽  
Bacterial Communities ◽  
Limit Of Detection ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Gene ◽  
Cycle Number ◽  
Intergenic Spacer Region ◽  
Automated Method ◽  
Ribosomal Intergenic Spacer

ABSTRACT An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Sequence Length Variation of Internal Genic Space of 16S rDNA-23S rDNA in Bacterium with High Yield of Hydrogen Production

2011 ◽  
Vol 183-185 ◽  
pp. 1413-1416
Author(s):  
Yong Feng Li ◽  
Yi Xuan Wang ◽  
Lu Wang ◽  
Zhan Qing Wang
Keyword(s):  
16S Rdna ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
High Yield ◽  
23S Rrna ◽  
Sequence Length ◽  
Rrna Gene ◽  
Length Variation ◽  
Intergenic Spacer Region

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.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

2006 ◽  
Vol 106 (3) ◽  
pp. 297-306 ◽  
Author(s):  
A. Llorens ◽  
M.J. Hinojo ◽  
R. Mateo ◽  
M.T. González-Jaén ◽  
F.M. Valle-Algarra ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Fusarium Spp ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

scholarly journals The Western Progression of Lyme Disease: Infectious and NonclonalBorrelia burgdorferi Sensu LatoPopulations in Grand Forks County, North Dakota

2014 ◽  
Vol 81 (1) ◽  
pp. 48-58 ◽  
Author(s):  
Brandee L. Stone ◽  
Nathan M. Russart ◽  
Robert A. Gaultney ◽  
Angela M. Floden ◽  
Jefferson A. Vaughan ◽  
...  
Keyword(s):  
Lyme Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
North Dakota ◽  
Intergenic Spacer ◽  
Rrna Gene ◽  
Content Type ◽  
Intergenic Spacer Region ◽  
Grand Forks ◽  
The 16S Rrna Gene

ABSTRACTScant attention has been paid to Lyme disease,Borrelia burgdorferi,Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports ofB. burgdorferiandI. scapularisin North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified asB. burgdorferi sensu latothrough sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileTintergenic spacer region,flaB,ospA,ospC, andp66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected withB. burgdorferiisolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, andB. burgdorferiM3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larvalI. scapularisticks were able to acquireB. burgdorferiM3 from infected mice; M3 was maintained inI. scapularisduring the molt from larva to nymph; and further, M3 was transmitted from infectedI. scapularisnymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectiousB. burgdorferipopulations in eastern North Dakota.

Differentiation of group VIII Spiroplasma strains with sequences of the 16S–23S rDNA intergenic spacer region

2004 ◽  
Vol 50 (12) ◽  
pp. 1061-1067 ◽  
Author(s):  
Laura B Regassa ◽  
Kimberly M Stewart ◽  
April C Murphy ◽  
Frank E French ◽  
Tao Lin ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Analytical Models ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group Viii ◽  
Similarity Coefficients ◽  
Intergenic Spacer Region ◽  
Intergenic Sequences ◽  
The Stability ◽  
The 16S Rrna Gene

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S–23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S–23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.Key words: Mollicutes, Spiroplasma, phylogeny, Tabanidae.

Seeing is believing: Can biofilm diagnostics guide individualized therapy in endocarditis?

2020 ◽  
Author(s):  
Judith Kikhney ◽  
Laura Kursawe ◽  
Swb Eichinger ◽  
Walter Eichinger ◽  
Julia Schmidt ◽  
...  
Keyword(s):  
Antibiotic Therapy ◽  
Biofilm Formation ◽  
Heart Valves ◽  
Single Cells ◽  
Intergenic Spacer ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Intergenic Spacer Region ◽  
Causative Microorganism ◽  
The Impact

<p><strong>Introduction</strong></p> <p>In Infective Endocarditis (IE), early diagnosis of the causative microorganism is crucial for correct antibiotic therapy, which improves the patients’ outcome.</p> <p><strong>Objectives</strong></p> <p>We studies the impact of biofilm formation in IE samples.</p> <p><strong>Materials & methods</strong></p> <p>We used Fluorescence in situ Hybridization (FISH) combined with 16S rRNA-gene PCR and sequencing to visualize and identify the infectious agents in native as well as prosthetic valves and to study any biofilm formation. The signal intensity of the fluorescence-labelled FISH probes correlates to a high ribosome content of the bacteria indicating metabolic activity at the time point of surgery. We developed a spacer FISH assay for the detection of the 16S-23S intergenic spacer region that is only present in actively transcribing cells to detect the activity of bacterial cells more precisely on a single cell level.</p> <p><strong>Results</strong></p> <p>FISH visualized bacteria in the heart valves ranging from single cells to highly organized biofilms. Interestingly, we found FISH positive bacteria in culture negative samples and samples from patients under antibiotic therapy. Using the spacer FISH, we visualized positive microbial cells in heart valves of patients under adequate therapy. Preliminary data point to a correlation between the biofilm state and treatment inefficiency.</p> <p><strong>Conclusion</strong></p> <p>FISH/PCR not only allows timely identification of the pathogens in IE, but also biofilm-staging and visualization of the effect of antimicrobial therapy at time of surgery. The technique provides crucial information for successful targeted antibiotic therapy, and it might guide therapeutical decisions in relation to biofilm state in the future.</p>

Incorrect sequencing and taxon misidentification: an example in the Trichinella genus

2010 ◽  
Vol 84 (3) ◽  
pp. 336-339 ◽  
Author(s):  
G. Marucci ◽  
G. La Rosa ◽  
E. Pozio
Keyword(s):  
Intergenic Spacer ◽  
Morphological Characters ◽  
Internal Transcribed Spacers ◽  
5S Rrna ◽  
Lsu Rdna ◽  
Intergenic Spacer Region ◽  
Reference Centre ◽  
Polymerase Chain ◽  
Taxon Identification ◽  
Selection Of

AbstractMolecular analyses such as polymerase chain reaction (PCR) and sequencing are very useful for taxon identification, especially when morphological characters useful for identifying taxa are lacking. However, the use of molecular tools can be the source of taxon misidentification if they are not correctly applied and the results are not critically evaluated and compared with the literature and GenBank data. We describe a case of misidentification of a taxon of the genus Trichinella due to sequencing mistakes, lack of reference material and selection of a single molecular marker. A Trichinella sp. isolate from an Iranian wild boar (Sus scrofa) was identified as belonging to the Nearctic species Trichinella murrelli, through the molecular analysis of the 5S rRNA intergenic spacer region. A successive molecular identification of the same isolate was performed by the International Trichinella Reference Centre in Rome, Italy, using the 5S rRNA intergenic spacer region, the LSU rDNA expansion segment five, and the internal transcribed spacers 1 and 2. According to these analyses, the Iranian isolate belonged to Trichinella britovi, a Palaearctic species already described in Iran.

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