Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli

Abstract

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.

FIG. 1.

Amplification of LT and ST genes in separate LC-PCR assays. The graphs depict fluorescence at 640 and 705 nm versus cycle number for E. coli strains with various combinations of target genes. (A) LT genes. •, H10407 (LT I positive, ST Ia positive, ST Ib positive); □, F5176 (LT I positive, ST Ia negative, ST Ib negative); —, negative control. (B) ST genes. •, H10407 (LT I positive, ST Ia positive, ST Ib positive); ▪, TX1 (LT I negative, ST Ia negative, ST Ib positive); ×, C4046 (LT I negative, ST Ia negative, ST Ib positive); □, R554 (LT I negative, ST Ia negative, ST Ib positive); ○, F7682 (LT I negative, ST Ia positive, ST Ib negative); —, negative control.

FIG. 2.

(A) Melting curve analysis performed on amplification products of two different LT-positive isolates. Strain H10407 represents a typical LT I-positive isolate, and strain F51761 is an ETEC isolate harboring a variant LT I sequence. (B and C) Sequence alignments with amplicons of the two different LT I-genotypes, the sequence under GenBank accession number S60731, and sensor hybridization probe LT-HP-1 (B) or anchor hybridization probe LT-HP-2 (C). Sequence identity is indicated by dashes. The observed T_ms are indicated.

FIG. 3.

(A) Melting curve analysis performed on amplification products of four different ST-positive isolates, representative of the different ST genotypes observed in this study. Strain H10407 represents a typical ST Ia-positive and ST Ib-positive isolate, strain F7682 is an isolate harboring a variant ST Ia sequence, and strains C4046 and R554 are ST Ib-positive isolates harboring variant ST Ib sequences. (B and C) Sequence alignments with amplicons of the four different ST-genotypes; the sequences under GenBank accession numbers M25607 (ST Ia), M34916 (ST Ib), and M18345 (variant ST Ib); and ST Ia-specific sensor hybridization probe ST-HP-1a (B), ST Ib-specific sensor hybridization probe ST-HP-1b (B), or anchor hybridization probe ST-HP-2 (C). Sequence identity is indicated by dashes. The observed T_ms are indicated.