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Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic Escherichia coli
- Source :
- Journal of Clinical Microbiology; September 2004, Vol. 42 Issue: 9 p4092-4100, 9p
- Publication Year :
- 2004
-
Abstract
- ABSTRACTTo 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. coliisolates 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.
Details
- Language :
- English
- ISSN :
- 00951137 and 1098660X
- Volume :
- 42
- Issue :
- 9
- Database :
- Supplemental Index
- Journal :
- Journal of Clinical Microbiology
- Publication Type :
- Periodical
- Accession number :
- ejs7914983
- Full Text :
- https://doi.org/10.1128/JCM.42.9.4092-4100.2004