16S–23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification

Mora, Diego; Ricci, Giovanni; Guglielmetti, Simone; Daffonchio, Daniele; Fortina, María Grazia

doi:10.1099/mic.0.25925-0

Cited by 48 publications

(31 citation statements)

References 15 publications

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“…This has been possible due to the high interand intraspecific variability (8,17,33,34,40). The advantage of this region is that the flanking 16S and 23S rRNA genes at the 5Ј and 3Ј ends of the ISR contain highly conserved sequences (i.e., the 16S and 23S rRNA genes).…”

Section: Discussionmentioning

confidence: 99%

Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori

et al. 2004

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The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

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Section: Discussionmentioning

confidence: 99%

Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori

et al. 2004

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“…Indeed, groESL gene-based techniques were restricted to the genus Streptococcus (49) and two different systems based on sequence of the sodA gene have been developped for the genera Streptococcus (35) and Enterococcus (36). Likewise, PCR-restriction fragment length polymorphism analysis of the entire 16S-23S rRNA region (45) included 178 strains belonging to 30 species and subspecies of the genus Streptococcus and further studies based on the analysis of the 16S-23S rRNA intergenic spacer included some species belonging to the same Streptococcus group, such as the S. mitis group (2), S. agalactiae group (6,15), and the S. thermophilus group (32). The rpoB gene-based primer pair determined in the present study may be helpful for the accurate detection and identification of Streptococcus species and related genera of medical interest.…”

Section: Discussionmentioning

confidence: 99%

rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus , Enterococcus , Gemella , Abiotrophia , and Granulicatella

et al. 2004

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We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair.

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“…An IGS region-specific primer pair was also used to compare the sequence variations within the ITS region. To analyze the variations in nucleotide sequences, direct sequencing of the amplified product, restriction fragment length polymorphism analysis (21), temperature gradient gel electrophoresis, denaturing gradient gel electrophoresis, amplified rRNA gene restriction analysis for the ribosomal gene, and SSCP analysis (9,(15)(16)(17) are being used by various workers. Among all these techniques, SSCP analysis is one of the most accurate and is often used for mutational studies and the detection of single nucleotide polymorphisms (12).…”

Section: Discussionmentioning

confidence: 99%

“…Besides this, intergenic spacer (IGS) region I (IGSI) and IGSII are found between the end of the LSU and start of the next SSU sequence (24). Many workers (9,(15)(16)(17) have used the single-stranded conformation polymorphism (SSCP) technique to identify sequence variations in a single strand of DNA due to its adoption to a unique conformation under nondenaturing conditions (12). Here we report on the experimental proof and the clinical laboratory diagnosis of three cases of corneal ulcer by PCR by the ITS SSCP technique, in which useful vision could be restored due to prompt diagnosis and specific antifungal therapy.…”

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confidence: 99%

Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis

Kumar

Shukla

2005

J Clin Microbiol

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The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccard's coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by Aspergillus fumigatus and was used for the diagnosis of fungal corneal ulcers in patients.Mycotic keratitis or fungal corneal infections have a worldwide distribution, and the incidence is higher in tropical and subtropical countries (8). More than 105 species of fungi belonging to 56 genera have been reported to cause oculomycosis. However, species of Fusarium, Aspergillus, Candida, and other hyaline and dematiaceous hyphomycetes are the usual isolates from patients with mycotic keratitis (20). The management of keratomycosis depends on rapid identification of the causal agent. The diagnosis is often delayed because of the poor availability of infected material from the cornea and the slow growth of a large number of fungi in routinely used culture media, and therefore, early intervention is not always possible and the patient's vision is often lost. In general, the diagnosis of fungal corneal ulcer is dependent on Gram and Giemsa staining, which have low sensitivities of about 50 to 80% (4). Recent advances in molecular biology techniques have opened the door for culture-independent diagnostic methods. Immunological detection (13) and identification by use of distinctive metabolites (2) and nucleic acid probes (7,25) are the tools most often used for diagnosis. One such technique is PCR, which has been shown to be useful for the culture-independent diagnosis of various microbial infections, including mycoses (10,11,22). To date, a few cases of mycotic keratitis have successfully been diagnosed by PCR (5, 6).Ribosomal DNA is the most conserved region in the genome, with capabilities of phylogenetic divergence (14). The whole rRNA gene contains a small subunit (SSU) 18S rRNA, 5.8S rRNA, and a large subunit (LSU) 28S rRNA. Internal transcribed spacer (ITS) region I (ITSI) and ITSII are more variable than the rest of the ribosomal gene subunits and are found between SSU rRNA and 5.8S ...

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16S–23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification

Cited by 48 publications

References 15 publications

Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori

Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori

rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus , Enterococcus , Gemella , Abiotrophia , and Granulicatella

Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis

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