|Home | About | Journals | Submit | Contact Us | Français|
Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx2). Our results showed that as few as 10 copies of stx2 could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx2 gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.
Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains have emerged as significant food-borne pathogens since their early identification in 1982 (7). They can cause severe clinical manifestations, including bloody diarrhea, hemorrhagic colitis, and postinfection hemolytic-uremic syndrome, symptoms associated with high morbidity and mortality. Cytotoxins, Shiga toxin types 1 and 2, produced by E. coli O157:H7 and STEC are responsible for these clinical symptoms (6). Infection with E. coli O157:H7 and STEC can occur sporadically, in small clusters, or in large outbreaks. The bacteria may be transmitted in a variety of ways, most commonly through food and water. Ruminants have been established as important reservoirs of E. coli O157:H7, and consequently, foods derived from or contaminated by these animals and their products are the major vehicles of transmission (5).
A number of methods have been developed for detecting the pathogens in food and clinical specimens, including culture isolation using selective media, such as sorbitol-substituted MacConkey agar and methylumbelliferyl-β-d-glucuronide agar, serological tests to detect O157 and H7 antigens, and immunological detection of Shiga toxins (5). To achieve sensitive, specific, and rapid detection of STEC and E. coli O157:H7 strains in clinical specimens and food products, several research teams have employed the PCR technique (1, 2). However, a number of drawbacks associated with such a PCR approach have limited its routine use in many laboratories (2).
We have recently developed a novel isothermal DNA amplification technology, termed ramification amplification or RAM (8). In this study, we developed a detection assay by combining magnetic bead-based DNA isolation, DNA amplification by RAM, and real-time fluorescence detection (9). The technique uses a circularizable probe to detect the target with subsequent amplification of the circular probe generated by a target-dependent ligation through a mechanism of primer extension, strand displacement, and ramification to achieve a billionfold amplification under isothermal conditions (Fig. (Fig.1)1) (11). The objective of this study was to determine the analytical sensitivity and specificity of the RAM assay for detecting the Shiga toxin 2 gene (stx2) and its feasibility for detecting E. coli O157:H7 and other STEC strains isolated from food and human specimens.
Bacterial isolates were obtained from the University of Maryland (18 isolates) and Center for Disease Control, China (12 isolates). All E. coli isolates were characterized by culture on sorbitol-substituted MacConkey agar and serologically typed for O and H antigens (Table (Table1).1). The presence of Shiga toxin genes (stx1 and stx2) was determined by PCR for all isolates. Of the 32 E. coli isolates, 23 were sorbitol negative and 22 were serologically determined to be E. coli O157:H7. Seven isolates were serologically determined to be non-O157 strains, of which six were sorbitol fermenters and only one was a nonfermenter. Three nonpathogenic E. coli isolates and one Shigella dysenteriae isolate obtained from the Clinical Microbiology Laboratory, Mount Sinai Hospital, were included as controls in this study.
The bacteria were inoculated onto a blood agar plate and incubated at 37°C overnight. A single colony was picked and suspended in water in a centrifuge tube. For the RAM assay, the bacteria were washed twice with saline and lysed in 100 μl of 5 M guanidium thiocyanate (GTC; Sigma, St. Louis, MO), 0.5% bovine serum albumin (Sigma), 80 mM EDTA, 400 mM Tris-HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine (Sigma) (9). The lysates were incubated at 100°C for 10 min and then at 60°C overnight. The lysed specimens were stored at −20°C until later use. For PCR assay, the bacteria were resuspended in 200 μl of distilled water, heated to 99°C for 10 min, and then centrifuged for 2 min at 12,000 rpm in an Eppendorf centrifuge. The resulting supernatant was used for PCR assay.
For quantitative analysis of E. coli O157:H7, a bacterial colony was picked and dissolved in saline. The bacterial density was determined by densitometry, and the concentration was determined by comparing the optical density value with those of standards of known bacterial concentrations. The bacteria were diluted with saline in a series of 10-fold dilutions, starting from 105 to 10 bacteria/μl. One microliter of suspension was inoculated onto MacConkey agar, and the number of colonies formed was determined to confirm the number of bacteria in initial dilutions. The supernatant was removed, and 5 M GTC was added to each tube, boiled at 100°C for 10 min, and incubated at 60°C overnight.
PCR was carried out in a 50-μl reaction mixture composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM concentrations of each deoxynucleoside triphosphate, 10 pmol of each primer (Table (Table2),2), 1 unit of Taq DNA polymerase (Roche, Indianapolis, IN), and 10 μl of bacterial lysate (2). The reaction was performed in a thermal cycler (GeneAmp 2700 PCR system; Perkin Elmer, Boston, MA) for 30 cycles of 94°C, 55°C, and 72°C for 1 min each. Fifteen microliters of PCR products was analyzed by gel electrophoresis using a 2.0% agarose gel and visualized after staining with 0.5 μg/ml of ethidium bromide. The expected PCR products are 614 bp for stx1 and 779 bp for stx2.
The RAM assay consists of steps, including hybridization of C-probe and capture probe to a target, capture of the hybrid onto magnetic beads, washing of the beads to remove unbound probes and cellular components, ligation of the 3′ and 5′ ends to form a closed C-probe, and amplification by primer extension, strand displacement, and ramification (Fig. (Fig.1)1) (8). Hybridization of C-probes and capture probes to targets was carried out in an 80-μl reaction mixture containing 2 M GTC, 0.5% bovine serum albumin (Sigma), 80 mM EDTA, 40 mM Tris-HCl (pH 7.5), 0.5% sodium-N-lauroylsarcosine (Sigma), 50 nM phosphorylated C-probe (Table (Table2),2), 2 μM capture probes, and 2 μl of lysed specimens or synthetic target DNA. The reaction mixture was incubated at 55°C for 2 h to allow complete hybridization. Then, 2 μl of magnetic beads (10 mg/ml; Dynal, Lake Success, NY) in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl was added, and the mixture was incubated to allow the beads coated with streptavidin to capture the biotin on the capture probe. The beads with bound complex were then washed twice with 400 μl of TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) at room temperature to remove unhybridized C-probes and other cellular components. Twenty microliters of ligase mixture containing 20 mM Tris-HCl (pH 7.6), 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM NAD, 0.1% Triton X-100, and 12 units of Taq DNA ligase (New England Biolabs, Boston, MA) was added to the bead pellet and incubated at 60°C for 10 min. Ligation of the 3′ and 5′ ends of the C-probe produced a closed circular DNA that was locked onto the target. After ligation, the RAM reaction was initiated by adding 50 μl of RAM reaction mixture containing 20 mM Tris-HCl buffer (pH 8.8), 300 μM deoxynucleoside triphosphate, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 1.2 μM concentrations of each forward and reverse primer (Table (Table2),2), 6.4 units of Bst DNA polymerase large fragment (New England Biolabs), 4 ng T4 Gene 32 protein (USB Biochemicals, Cleveland, OH), and 6% dimethyl sulfoxide (Sigma). This mixture was then incubated at 63°C for 1 h to start the ramification reaction, followed by heating at 95°C for 5 min to inactivate the Bst DNA polymerase. Fifteen microliters of the RAM products was transferred to a 5-μl mixture containing 50 mM Tris-HCl (pH 7), 15 units of EcoRI (Boehringer Mannheim, Mannheim, Germany), 100 mM MgCl2, and 1 mM dithioerythritol, and the reaction mixture was incubated at 37°C for 3 h. Fifteen microliters of the digested products was analyzed on a 2% agarose gel.
The RAM reactions were carried out as described above except that 2.5 μl of 1:5,000 diluted SYBR Green I (Roche) was added to each reaction mixture. SYBR Green I is a fluorochrome that, upon binding to the minor groove of double-stranded DNA, emits an intense green fluorescent signal which can be readily detected using a fluorometer. The reactions were monitored at 37°C for 2 h in a SmartCycler (Cepheid, Sunnyvale, CA).
RAM technology employs a circular probe for target detection and amplification, which offers several unique features (Fig. (Fig.1)1) (10, 11). The formation of a closed C-probe requires target-specific ligation of the C-probe; the 5′ end of the C-probe must align perfectly with its 3′ end on the target DNA for ligation to occur (3, 4). The C-probe can then be amplified with a set of generic primers that bind to the loop region of the C-probe (3), achieving an exponential amplification with a power similar to that of PCR. However, since no temperature cycling is required, the reaction can be carried out at a constant temperature, obviating the use of an expensive thermocycler.
We initially determined the analytical sensitivity of the RAM assay using a synthetic stx2 DNA target (Table (Table2).2). The DNA was diluted in 100-fold serial dilutions from 105 to 103 to 101 molecules/2 μl and was used to initiate the RAM reaction. The lowest number of targets detected by RAM assay was 10 molecules (Fig. (Fig.2A),2A), and the reactions were confirmed by finding the correct products (124 bp) in each lane after digestion with EcoRI, establishing the C-probe as their source. In the absence of a target molecule, no DNA was produced, validating a target-dependent amplification of the C-probe. The assay sensitivity was further determined using an E. coli O157:H7 strain. The bacterial concentration was determined by densitometry. The bacteria were lysed in 5 M GTC, diluted to 105, 103, and 101 copies/2 μl, and then used to initiate the RAM reaction. The results in Fig. Fig.2B2B showed that the assay was able to detect as few as 10 bacteria, a sensitivity comparable to that of PCR.
To determine the assay specificity, we tested several bacterial strains, including E. coli O157:H7, E. coli O46:H38, E. coli O111:NM, three nonpathogenic E. coli isolates, and S. dysenteriae by RAM assay to determine the assay specificity. As expected, E. coli O157:H7, E. coli O46:H38, and E. coli O111:NM were positive for the stx2 gene, while S. dysenteriae and the three nonpathogenic E. coli isolates were negative (Fig. (Fig.3).3). These results evidently confirmed the specificity of the RAM assay.
We then tested 29 pathogenic E. coli isolates from human and food samples for the presence of stx genes (Table (Table1).1). The presence of stx genes was determined by PCR using primers specific for stx1 and stx2 (Table (Table2).2). Since the C-probe was designed specifically to recognize stx2, it was expected that the presence of stx2 would give a positive result by RAM assay. All 27 Shiga toxin 2-producing E. coli isolates were positive by RAM assay, irrespective of their serological types (Table (Table1).1). Two pathogenic E. coli isolates containing only stx1 were negative by RAM assay, thus confirming the specificity of the C-probe.
It will be desirable to detect the RAM reaction by real-time monitoring instead of gel electrophoresis. We applied SYBR Green I dye in our RAM reactions. The initial experiment was performed using a lysed E. coli O157:H7 sample. Our results showed that as few as 10 bacteria could be detected and that the time needed for the emergence of a detectable signal was dependent on the target concentration (Fig. (Fig.4A).4A). Additionally, we have employed this method for the detection of bacterial isolates, and Fig. Fig.4B4B shows an example of real-time RAM assay of three isolates. This study showed that real-time RAM assay can be developed for diagnostic use, which can significantly shorten the assay time and eliminate the possibility of carryover contamination.
This study demonstrated that RAM assay could be another DNA amplification method to detect STEC. The high sensitivity and specificity of the RAM assay coupled with its ease of application encourage further investigation and improvement of this technique. Future study will focus on designing several C-probes to target other virulence genes, such as the stx1, hly, and eae genes, for multiplex RAM assay. We also hope, in the near future, to conduct a larger clinical study to determine assay sensitivity and specificity for complex clinical samples such as stool and food.
This work was supported in part by grants from the United States Department of Agriculture (2003-35201-12856) and the National Natural Science Foundation of China (30271251).