RAM technology employs a circular probe for target detection and amplification, which offers several unique features (Fig. ) (10
). 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
). 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 ). 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. ), 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. showed that the assay was able to detect as few as 10 bacteria, a sensitivity comparable to that of PCR.
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 (more ...)
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. ). These results evidently confirmed the specificity of the RAM assay.
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 (more ...)
We then tested 29 pathogenic E. coli isolates from human and food samples for the presence of stx genes (Table ). The presence of stx genes was determined by PCR using primers specific for stx1 and stx2 (Table ). 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 ). 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. ). Additionally, we have employed this method for the detection of bacterial isolates, and Fig. 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.
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 (more ...)
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.