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Journal of Clinical Microbiology, December 2005, p. 6086-6090, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6086-6090.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Ramification Amplification Assay for Detection of Escherichia coli O157:H7 and Other E. coli Shiga Toxin-Producing Strains

Fan Li,¹ Chunyan Zhao,¹ Wandi Zhang,² Shenghui Cui,³ Jianghong Meng,³ Josephine Wu,² and David Y. Zhang^2*

Department of Pathogenobiology, School of Basic Medical Sciences, Jilin University, Changchun, China,¹ Department of Pathology, Mount Sinai School of Medicine, New York, New York,² Department of Nutrition and Food Science, University of Maryland, College Park, Maryland³

Received 10 February 2005/ Returned for modification 28 April 2005/ Accepted 17 August 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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-ß-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. 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.

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FIG. 1. Schematic representation of RAM assay. Target DNA, capture probe, C-probe, and paramagnetic bead are added to hybridization buffer to allow the formation of a hybrid complex. The hybrid is captured on a paramagnetic bead, allowing extensive washing to remove unbound C-probe and cellular components. The C-probe aligned on the target is linked together by a DNA ligase. RAM amplification is then carried out by the addition of forward () and reverse () primers and DNA polymerase (•). The forward primer bound to the C-probe is extended by the polymerase and continues after one round of synthesis by displacing the bound forward primer and its extended product, generating a long single-stranded DNA (ssDNA) with repeated sequence. With the reactions taking place, multiple reverse primers can bind to the nascent ssDNA as their binding sites become available. Each bound reverse primer will be extended and displace the downstream primers and their extended products. The forward primer binding sites of the displaced ssDNA are then available for the forward primer to bind and extend in a similar fashion, thus forming a large ramifying DNA complex. Finally, the RAM products are examined by gel electrophoresis after EcoRI digestion. (Inset) The C-probe hybridizes to the target through their complementary regions (f and g), and the sequence at the noncomplementary region (e) is generic for the binding of primers. The C-probe-target hybrid is captured on a paramagnetic bead (a) through the binding of the biotin moiety (c) on the capture probe (d) to the streptavidin (b) that the beads were coated with.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Sample preparation. 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 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.

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TABLE 1. Characteristics and genotyping of bacterial isolates by PCR and RAM

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 assay. 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 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.

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TABLE 2. Sequences of circular probe, capture probe, RAM and PCR primers, and synthetic target sequence

RAM assay. 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. 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 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 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.

Real-time RAM. 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).

Top Abstract Introduction Materials and Methods Results and Discussion References

 
RAM technology employs a circular probe for target detection and amplification, which offers several unique features (Fig. 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 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. 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. 2B showed that the assay was able to detect as few as 10 bacteria, a sensitivity comparable to that of PCR.

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FIG. 2. Sensitivity of RAM assay for detecting Shiga toxin target. (A) The RAM reactions were initiated with the synthetic Shiga toxin 2 targets of 105, 103, and 10 copies. The RAM products were examined on a 2% agarose gel following EcoRI digestion. The results showed that 10 copies of Shiga toxin 2 targets were detected. Lane 0, no target present; lane M, molecular marker (pBR 322/MspI; New England Biolabs). (B) Sensitivity was confirmed using E. coli O157:H7 that was diluted to 105, 103, and 10 copies. As few as 10 bacteria of E. coli O157:H7 can be detected. Lane 0, no target present; lane M, molecular marker (pBR 322/MspI; New England Biolabs).

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. 3). These results evidently confirmed the specificity of the RAM assay.

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FIG. 3. Specificity of RAM assay for detecting Shiga toxin genes in bacteria. Seven bacterial isolates were tested with the RAM assay. Our results showed that the Shiga toxin 2 gene was present only in pathogenic E. coli. Lane 1, E. coli O157:H7; lanes 2 to 4, nonpathogenic E. coli; lane 5, E. coli O46:H38; lane 6, E. coli O111:NM; lane 7, S. dysenteriae.

We then tested 29 pathogenic E. coli isolates from human and food samples for the presence of stx genes (Table 1). The presence of stx genes was determined by PCR using primers specific for stx₁ and stx₂ (Table 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 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. 4A). Additionally, we have employed this method for the detection of bacterial isolates, and Fig. 4B 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.

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FIG. 4. Real-time RAM assay. (A) RAM assays were initiated with 107, 105, 103, and 10 cells of E. coli O157:H7 in the presence of SYBR Green I; (B) RAM reactions were monitored using a SmartCycler. Three isolates were assayed with RAM in the presence of SYBR Green I, and RAM products were detected as the intensity of the fluorescent signal increased.

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).

 
* Corresponding author. Mailing address: Molecular Pathology Laboratory, Mount Sinai School of Medicine, Box 1122, One Gustave Levy Place, Box 1122, New York, NY 10029. Phone: (212) 659-8173. Fax: (212) 427-2082. E-mail: David.Zhang{at}mssm.edu.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Journal of Clinical Microbiology, December 2005, p. 6086-6090, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6086-6090.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hussein, H. S. (2007). Prevalence and pathogenicity of Shiga toxin-producing Escherichia coli in beef cattle and their products. J ANIM SCI 85: E63-E72 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, F. Articles by Zhang, D. Y. Search for Related Content PubMed PubMed Citation Articles by Li, F. Articles by Zhang, D. Y.