Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples

Abstract

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

Figure 1.

Aligned enterotoxigenic Escherichia coli (ETEC) sta sequences. Sequences were aligned using Clustal X and then shaded using Boxshade 3.21 (www.ch.embnet.org). The sta1- and sta2-specific primers used for real-time quantitative polymerase chain reaction (qPCR) are indicated by arrows. GenBank accession nos. are as follows: pETEC666, GI 309705521; pTE503, GI 147877; pTC, GI 356598343; pST18, GI 145860; ESF0041, GI 43704; pETEC948, GI 309706192; 100 kb plasmid, GI 82697140; strain CRL 25090, GI 145862; 150 kb plasmid, GI 148029; pYK007, GI 147875; strain 153837-2, GI 146407.

Figure 2.

Representative standard curves for each toxin-specific quantitative real-time polymerase chain reaction assay: (A) eltA; (B) sta1; (C) sta2. Cycle threshold (Cq) is plotted versus log₁₀(plasmid molecules) per reaction.

Figure 3.

Effect of dilution of stool DNA on eltA quantitative real-time polymerase chain reaction accuracy for samples inoculated with serial dilutions of H10407 cells ranging from 10⁹ to 10² cells/700 mg aliquot. Black bar: theoretical gene copy number per reaction. Assayed gene quantity using undiluted DNA (light gray bars), 1:10 stool DNA (white bars), and 1:100 stool DNA (dark gray bars) plotted for each stool sample, where the concentration of the H10407 inoculum (cells/sample) is indicated in parentheses. Each assay was performed at least twice; error bars represent the standard deviation.

Figure 4.

DNA hybridization filter showing results for 10 Escherichia coli colonies isolated from stool samples inoculated with 10⁷, 10⁸, or 10⁹ H10407 cells and probed with end-labeled primers specific for heat stable enterotoxin (ST, left) and heat labile enterotoxin (LT, right) genes. (A) Hybridization membrane, hybridized first with the ST probes and then washed and reprobed with the LT probe; (B) key showing the location of individual spotted colonies, interpretation of results, and location of negative (E. coli NCTC 9001) and positive (E. coli H10407) control colonies. No positive signals were observed at dilutions below 10⁷ H10407 cells per stool sample (filters not shown).

Figure 5.

RNA structures of stem II and loop II for plasmids pETEC948 and pETEC666 predicted using mfold version 3.5. Base positions relative to the 5′ start of the complete RNA molecule are indicated, and the calculated free energies (kcal/mol) of the stem-loop structure alone are shown below the structure. The 5′ stem I and loop I sequences are not shown to simplify presentation.