Genomic fingerprinting ofFrankiamicrosymbionts fromCeanothuscopopulations using repetitive sequences and polymerase chain reactions

1999 ◽  
Vol 77 (9) ◽  
pp. 1220-1230 ◽  
Author(s):  
Soon-Chun Jeong ◽  
David D Myrold
Keyword(s):  
Dna Sequencing ◽  
Dna Analysis ◽  
Repetitive Sequences ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Genomic Fingerprinting ◽  
Chain Reactions ◽  
Intergenic Spacer Region ◽  
Polymerase Chain

Specificity between Ceanothus species and their microsymbionts, Frankia, were investigated with nodules collected from three geographically separated copopulations of Ceanothus species. Nodules were analyzed using DNA sequencing and repetitive sequence polymerase chain reaction (rep-PCR) techniques. DNA sequencing of the intergenic spacer region between 16S and 23S rRNA genes suggested that Ceanothus-microsymbiotic Frankia are closely related at the intraspecific level. Diversity of the microsymbionts was further analyzed by genomic fingerprinting using repetitive sequences and PCR. A newly designed direct repeat (DR) sequence and a BOX sequence were used as PCR primers after justification that these primers can generate Frankia-specific fingerprints from nodule DNA. Analysis of the nodules using BOX- and DR-PCR showed that Ceanothus-microsymbiotic Frankia exhibited less diversity within each copopulation than among copopulations. These data suggested that geographic separation plays a more important role for divergence of Ceanothus-microsymbiotic Frankia than host plant.Key words: Frankia, Ceanothus, rep-PCR, diversity.

Detection of phytoplasmas in dieback, yellow crinkle, and mosaic diseases of papaya using polymerase chain reaction techniques

1996 ◽  
Vol 47 (3) ◽  
pp. 387 ◽  
Author(s):  
B Liu ◽  
DT White ◽  
KB Walsh ◽  
PT Scott
Keyword(s):  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Pcr Product ◽  
Polymerase Chain ◽  
23S Rrna Genes ◽  
The 16S Rrna Gene

Oligonucleotide primers complementary to regions specific to plant-pathogenic mycoplasma-like organisms (phytoplasmas) were used in polymerase chain reactions on tissue samples from dieback, yellow crinkle, and mosaic affected papaya plants. The primer pair P068/P069, which hybridise to internal regions of the 16s rRNA gene, amplified an approximately 560 bp product in dieback, yellow crinkle and mosaic affected papaya. The primer pair P3/P7, which hybridise to the spacer region between the 16s and 23s rRNA genes, amplified an approximately 300 bp fragment in yellow crinkle and mosaic affected papaya, with no product from dieback affected plants. No PCR product was obtained with either set of primers from healthy plants. An identical Alu I restriction enzyme profile was obtained with all three 560 bp products. This study provides the first evidence for the association of phytoplasmas with papaya mosaic and Australian papaya dieback.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1239-1247 ◽  
Author(s):  
Christine Yeates ◽  
Aaron M. Saunders ◽  
Gregory R. Crocetti ◽  
Linda L. Blackall
Keyword(s):  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Target Region ◽  
Intergenic Spacer Region ◽  
Probe Target ◽  
Pcr Products ◽  
23S Rrna Genes

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium ‘Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027–1043 in the 23S rRNA and differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in ‘Candidatus C. phosphatis’. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as ‘Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of ‘Candidatus C. phosphatis' with G at position 1033 and GAM42a (G–A) or BET42a (G–T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some ‘Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to ‘Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

Rapid Polymerase Chain Reaction/DNA Probe Membrane-Based Assay for the Detection of Listeria and Listeria monocytogenes in Food

2000 ◽  
Vol 63 (3) ◽  
pp. 337-342 ◽  
Author(s):  
L. O'CONNOR ◽  
J. JOY ◽  
M. KANE ◽  
T. SMITH ◽  
M. MAHER
Keyword(s):  
Polymerase Chain Reaction ◽  
Microbiological Quality ◽  
Intergenic Spacer ◽  
Pcr Primers ◽  
Dna Probe ◽  
23S Rrna ◽  
Chain Reaction ◽  
Intergenic Spacer Region ◽  
Rapid Polymerase Chain Reaction ◽  
Polymerase Chain

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.

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 Sequencing of the Ribosomal Intergenic Spacer Region for Strain Identification of Porphyromonas gingivalis

1999 ◽  
Vol 37 (8) ◽  
pp. 2723-2725 ◽  
Author(s):  
Robert W. Rumpf ◽  
Ann L. Griffen ◽  
Bo-Gui Wen ◽  
Eugene J. Leys
Keyword(s):  
Porphyromonas Gingivalis ◽  
Intergenic Spacer ◽  
Variable Region ◽  
Rrna Genes ◽  
23S Rrna ◽  
Clinical Samples ◽  
Strain Identification ◽  
Intergenic Spacer Region ◽  
Ribosomal Intergenic Spacer ◽  
23S Rrna Genes

The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivaliswere amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalisISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison.

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.

scholarly journals Diversity of Salmonella Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions

2000 ◽  
Vol 66 (4) ◽  
pp. 1544-1552 ◽  
Author(s):  
Julia Baudart ◽  
Karine Lemarchand ◽  
Anne Brisabois ◽  
Philippe Lebaron
Keyword(s):  
Pathogenic Bacteria ◽  
Natural Waters ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Natural Environments ◽  
Storm Events ◽  
Intergenic Spacer Region ◽  
Coastal Watershed

ABSTRACT Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater.Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonellastrains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.

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