RESUMO
Rapid molecular diagnostic testing for respiratory infections can improve patient care and minimize unnecessary prescriptions of antibiotics. We present the preliminary clinical evaluation of Orion GenRead® RSV, a novel, rapid, and easy-to-use molecular test for the diagnosis of respiratory syncytial virus (RSV) infection. The sensitivity and specificity of Orion GenRead RSV were 99% and 100%, respectively. Orion GenRead RSV detected RSV-positive specimens within 15â¯min. The performance of Orion GenRead RSV was similar to that of the reference method and this test could rapidly detect RSV within minutes. Orion GenRead RSV is applicable for near-patient testing.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/virologia , Infecções Respiratórias/virologia , Fatores de TempoRESUMO
We report a multiplatform real-time polymerase chain reaction methodology based on genes encoding for the regulatory toxR activator and enterotoxin A protein to determine enterotoxigenic Vibrio cholerae types from other vibrios. This assay, which was successfully validated on a collection of 87 bacterial strains, including 63 representatives of V. cholerae and 8 noncholera vibrios provides a rapid tool for detection and identification of cholera.
Assuntos
Reação em Cadeia da Polimerase/métodos , Vibrio cholerae/isolamento & purificação , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Cólera/diagnóstico , Cólera/microbiologia , Proteínas de Ligação a DNA/genética , Enterotoxinas/genética , Microbiologia Ambiental , Humanos , Sensibilidade e Especificidade , Fatores de Transcrição/genética , Vibrio cholerae/classificação , Vibrio cholerae/genéticaRESUMO
A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.
Assuntos
Peste/diagnóstico , Reação em Cadeia da Polimerase/métodos , Yersinia pestis/isolamento & purificação , Infecções por Yersinia pseudotuberculosis/diagnóstico , Yersinia pseudotuberculosis/isolamento & purificação , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Bioterrorismo , Primers do DNA , DNA Bacteriano/análise , Genes Bacterianos , Humanos , Metiltransferases/genética , Dados de Sequência Molecular , Ativadores de Plasminogênio/genética , Sensibilidade e Especificidade , Alinhamento de Sequência , Yersinia pestis/genética , Yersinia pseudotuberculosis/genéticaRESUMO
BACKGROUND: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. METHODS: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. RESULTS: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. CONCLUSIONS: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.
