RESUMO
We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy.
Assuntos
Primers do DNA/metabolismo , Fluoresceínas/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Etídio/metabolismo , Corantes Fluorescentes/metabolismo , Substâncias Intercalantes/metabolismo , Sensibilidade e Especificidade , Escherichia coli Shiga Toxigênica/citologia , Escherichia coli Shiga Toxigênica/genética , Coloração e Rotulagem , Temperatura , Fatores de TempoRESUMO
The serotyping of staphylocoagulase is widely used in Japan. However, the conventional immunoassay based on neutralization of the antisera is so laborious and time-consuming that it is not widely used in the other countries. In order to overcome these drawbacks we developed a novel staphylocoagulase serotyping method based on a microplate format using polystyrene latex particles. Addition of latex particles promotes the formation of fibrin complexes, which represents a more rapidly and easily detected endpoint. For 83 strains, 90% were classified into serotypes within 3 h, and there was no discrepancy in the results between our method and the conventional method. These results indicate that the present microplate method is rapid, simple, and interpretable.
Assuntos
Coagulase/isolamento & purificação , Sorotipagem/métodos , Microesferas , Poliestirenos , Staphylococcus aureus/enzimologiaRESUMO
We developed a novel PCR-based method for coagulase serotyping. Coagulase gene amplicons biotinylated by PCR were identified by microplate hybridization (MPH) using serotype-specific probes. The conventional serotyping method, which is strongly dependent on coagulase activity, may sometimes give a mistaken determination of the serotype, especially in cases where there is high coagulase activity. In contrast, the results of PCR-MPH are not affected by coagulase activity. Furthermore, once the isolated colonies are obtained, it only takes 3 h to perform PCR-MPH, and the interpretation of the results is entirely objective. We compared PCR-MPH with the conventional method for 90 strains of coagulase-producing Staphylococcus aureus. The same results were found for both the PCR-MPH and conventional methods, and thus, our results indicate that PCR-MPH is a rapid, objective, and reliable method for coagulase serotyping.
