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1.
J Clin Microbiol ; 42(9): 4092-100, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15364995

RESUMO

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Enterotoxinas/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/isolamento & purificação , Toxinas Bacterianas/genética , Sequência de Bases , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Enterotoxinas/genética , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Variação Genética , Humanos , Reação em Cadeia da Polimerase/métodos , Sorotipagem
2.
J Clin Microbiol ; 40(7): 2555-65, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12089277

RESUMO

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.


Assuntos
Adesinas Bacterianas/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Hemolisinas/genética , Reação em Cadeia da Polimerase/métodos , Toxina Shiga/genética , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Corantes Fluorescentes , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Toxina Shiga/biossíntese
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