Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli.
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.
Reischl
Udo
U
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany. Udo.Reischl@klinik.uni-regensburg.de
Youssef
Mohammad T
MT
Wolf
Hans
H
Hyytia-Trees
Eija
E
Strockbine
Nancy A
NA
D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004768:Enterotoxins / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification*; D014644:Genetic Variation; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012703:Serotyping
D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); C027538:heat-labile enterotoxin, E coli