Extended Bibliography: |
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Ref #: |
55499 |
Author(s): |
Blaiotta,G.;Pennacchia,C.;Ercolini,D.;Moschetti,G.;Villani,F. |
Journal: |
Syst Appl Microbiol |
Title: |
Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages |
Volume: |
26 |
Page(s): |
423-33 |
Year: |
2003 |
Keyword(s): |
DNA, Bacterial/isolation & purification
*DNA, Ribosomal/analysis
*DNA, Ribosomal Spacer/analysis
Electrophoresis, Agar Gel
Electrophoresis, Polyacrylamide Gel
Food Microbiology
Gram-Positive Cocci/classification/genetics/isolation & purification
Meat Products/microbiology
Micrococcus
Molecular Sequence Data
Polymerase Chain Reaction
RNA, Ribosomal, 16S/genetics
RNA, Ribosomal, 23S/genetics
Staphylococcus/*classification/genetics/isolation & purification
|
Remarks: |
Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains. |
URL: |
14529185 |
|
Ref #: |
81199 |
Author(s): |
Becker,K.;Harmsen,D.;Mellmann,A.;Meier,C.;Schumann,P.;Peters,G.;von Eiff,C. |
Journal: |
J Clin Microbiol |
Title: |
Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species |
Volume: |
42 |
Page(s): |
4988-95 |
Year: |
2004 |
Keyword(s): |
GENBANK/AY688029
GENBANK/AY688030
GENBANK/AY688031
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GENBANK/AY688033
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GENBANK/AY688106
GENBANK/AY688107
GENBANK/AY688108
GENBANK/AY688109
*Bacterial Typing Techniques
DNA, Bacterial/analysis
DNA, Ribosomal/analysis
*Databases, Nucleic Acid
Genes, rRNA
Humans
Molecular Sequence Data
RNA, Ribosomal, 16S/*genetics
Sequence Analysis, DNA
Species Specificity
Staphylococcal Infections/*microbiology
Staphylococcus/*classification/genetics/metabolism
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Remarks: |
To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5' end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of > or =98.7% similarity for a "distinct species," a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMerieux, Marcy I'Etoile, France) and VITEK 2 (bioMerieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants. |
URL: |
15528685 |
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