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F Bekkaoui

Publications and source records attributed to F Bekkaoui.

6 recordsLinked to original sources
PubMed2000

Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.

Bacterial Proteins↗
PubMed1999

Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology.

A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at the 3'-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT-EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24-48 h required for detection of methicillin resistance by conventional susceptibility tests.

Bacterial Proteins↗
PubMed1998

Spermine-mediated improvement of cycling probe reaction.

Cycling probe technology (CPT) represents a simple method for detection of DNA target sequences. Cycling probe technology utilizes a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target. Probe cleavage in the presence or absence of target generates CPT product or background, respectively. Addition of non-homologous DNA into the CPT reaction affects the background and CPT product. Low amounts of human DNA (4-40 ng) result in high background while higher amounts (40-400 ng) inhibit the reaction. The simultaneous addition of spermine and EGTA into the CPT reaction containing human DNA resulted in a significant release of the inhibition and a reduction of background. The presence of spermine alone caused an increase of probe cleavage whereas addition of EGTA increased the specificity of the CPT. A possible mechanism by which spermine could lead to this improvement of CPT has been proposed. Using a membrane-binding assay, the authors demonstrated that human DNA competes with the probe for binding to RNase H. Furthermore, by using a DNA-agarose column, it has been shown that such RNase H-DNA binding can be disrupted by spermine. Within the CPT reaction, similar spermine-mediated displacement of RNase H from human DNA could lead to an improved CPT efficiency.

Aurora Kinases↗
PubMed1996

Cycling probe technology with RNase H attached to an oligonucleotide.

A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits. Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties. Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme. The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe. These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.

Bacterial Proteins↗
PubMed1993

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae.

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.

Acetolactate Synthase↗