Genetic Identification of <i>Staphylococcus aureus</i> by Polymerase Chain Reaction Using Single‐Base‐Pair Mismatch in 16S Ribosomal RNA Gene

Saruta, Katsutoshi; Hoshina, Sadayori; Machida, Katsuhiko

doi:10.1111/j.1348-0421.1995.tb03280.x

Cited by 30 publications

(26 citation statements)

References 19 publications

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“…Consequently, manifold molecular strategies have been developed to overcome the limitations of the traditional identification approaches. In addition to universal gene targets based on the ribosomal gene cluster or on the RNA polymerase B (rpoB) (2,29,31,34), several specific targets, such as the staphylococcal coagulase gene (coa), a thermostable nuclease gene (nuc), a superoxide dismutase gene (sodM), the HSP60 gene, specific genomic DNA fragments of unknown function, and the fem factor-encoding genes (6,13,24,26,32,37), were used for molecular detection and identification of S. aureus. However, some of these studies concentrated on only a limited number of staphylococcal species.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular methods represent alternatives combining high sensitivity, specificity, speed, and reliability for identification. Several nucleic acid amplification-based assays have been reported for S. aureus that are based on specific gene targets as well as universal sequences, offering different advantages and limitations (2,6,12,13,27,29,31,38,42). Thus, additional reliable tools for detection of S. aureus are of major interest.…”

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confidence: 99%

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eapGene as Novel Target for Specific Identification ofStaphylococcus aureus

et al. 2008

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The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n ‫؍‬ 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

eapGene as Novel Target for Specific Identification ofStaphylococcus aureus

et al. 2008

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“…Furthermore, commercially available panels, which are based on functional differences in metabolic pathways, do not allow a reliable distinction between different coagulase-negative staphylococci (CNS). A number of PCR-based methods for the species-specific detection of S. aureus, S. epidermidis, and S. saprophyticus have been reported (4,8,26,27,31,35). Regarding PCR assays for detection of staphylococci at the genus level, the 16S rRNA or the HSP60 genes have been used as targets (9,10).…”

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Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels

et al. 2001

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We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the tuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several tuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus demonstrated sufficient interspecies polymorphism to generate genus-and species-specific capture probes. This sequence information allowed the development of Staphylococcus-specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis, or S. saprophyticus) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.

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“…Although PCR ampli cation of bacterial DNA is rapid and sensitive, most current methods are too speci c to be used for initially evaluating the possibility of bacterial infections when the identity of the organism is unknown. The speci c sequence of the 16S ribosomal RNA (16S rRNA) gene was used recently to detect, identify and classify bacteria (5)(6)(7). This sequence was chosen for several reasons.…”

Section: Discussionmentioning

confidence: 99%

Detection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemia

Shang

Chen

2001

Acta Paediatrica

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Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram‐positive bacteria from gram‐negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram‐positive probe and a gram‐negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T‐12 g. All 26 culture‐positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram‐negative and gram‐positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates.

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Genetic Identification of Staphylococcus aureus by Polymerase Chain Reaction Using Single‐Base‐Pair Mismatch in 16S Ribosomal RNA Gene

Cited by 30 publications

References 19 publications

eapGene as Novel Target for Specific Identification ofStaphylococcus aureus

eapGene as Novel Target for Specific Identification ofStaphylococcus aureus

Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels

Detection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemia

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