Reischl, Udo and Youssef, Mohammad T. and Wolf, Hans and Hyytia-Trees, Eija and Strockbine, Nancy A. (2004) Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli. JOURNAL OF CLINICAL MICROBIOLOGY, 42 (9). pp. 4092-4100. ISSN 0095-1137,
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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 1) 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 1, ST la 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 la and ST 1b. 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | POLYMERASE-CHAIN-REACTION; RAPID IDENTIFICATION; TRAVELERS DIARRHEA; TOXIN; SEQUENCE; OUTBREAK; PROBES; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 30 Jun 2021 08:46 |
| Last Modified: | 30 Jun 2021 08:46 |
| URI: | https://pred.uni-regensburg.de/id/eprint/37266 |
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