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1.
Diagn Microbiol Infect Dis ; 34(2): 83-90, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10354856

RESUMO

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.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Genes Bacterianos , Hexosiltransferases , Resistência a Meticilina/genética , Técnicas de Sonda Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , Staphylococcus/genética , Colorimetria , Humanos , Técnicas Imunoenzimáticas , Meticilina/farmacologia , Proteínas de Ligação às Penicilinas , Penicilinas/farmacologia , Reação em Cadeia da Polimerase/métodos , Ribonuclease H/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus/efeitos dos fármacos
2.
J Clin Microbiol ; 38(7): 2525-9, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10878037

RESUMO

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.


Assuntos
Técnicas Bacteriológicas , Imunoensaio , Resistência a Meticilina/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/genética , Meios de Cultura , Sondas de DNA , Humanos , Reação em Cadeia da Polimerase , Sondas RNA , Fitas Reagentes , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
3.
Plant J ; 9(2): 137-45, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8820603

RESUMO

Plants with defects in the synthesis of their epicuticular wax layer, eceriferum mutants (cer), are readily detected by the naked eye as bright green glossy plants when compared with the more glaucous normal plants. In a previous report, evidence was presented for the isolation of three lines from the Arabidopsis thaliana transformant collection (BRL5, BRL7 and BRL9) which failed to complement the cer2 mutant isolated previously. The analysis of the chemical composition of the epicuticular wax of these mutants suggests that the cer2 mutant of Arabidopsis is defective in very long chain fatty acid elongation. This paper reports the molecular cloning of the CER2 gene of Arabidopsis through the isolation of plant DNA flanking the site of T-DNA insertion as well as the characterization of the two independent T-DNA insertion mutant alleles, BRL5 and BRL9, of this gene. In the mutant line BRL5, T-DNA was found to be inserted in the second exon of the CER2 gene whereas in BRL9, T-DNA is inserted in the only intron of this gene. Nucleotide sequence analysis suggests that the ORF encodes a 47.3 kDa polypeptide. High levels of CER2 transcripts were detected in stems and flowers. The predicted amino acid sequence of the CER2 gene product reveals little homology with known protein sequences. In accordance with structural characterization of the T-DNA insertion mutants, no evidence of transcripts derived from the CER2 gene was found in either BRL5 or BRL9.


Assuntos
Arabidopsis/genética , Genes de Plantas , Proteínas de Plantas/genética , Ceras/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Primers do DNA , Dados de Sequência Molecular , Mutagênese Insercional , Brotos de Planta/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Distribuição Tecidual
4.
Genome ; 36(3): 610-8, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18470011

RESUMO

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.

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