The maximum numbers of different cloned replicons that could be detected in one reaction depended on the temperature of disassociation. All primers sets showed a clear specific melting peak, although at concentrations lower than 5 fg additional aspecific peaks appeared. Because of the overlap of
disassociation temperatures we chose to amplify a maximum of 3 different replicons per reaction. Replicon typing of plasmids in wild type strains The same amplification procedure was used on the crude lysates of wild type (WT) strains to evaluate applicability in a routine setting. The wild type plasmids were analyzed in fresh crude bacterial lysates. The lysates were tested in a 10-1 to 10-9 dilution for each strain. Figure 1 illustrates an example of the results obtained with different buy 5-Fluoracil www.selleckchem.com/products/H-89-dihydrochloride.html concentrations of DNA of an E. coli containing replicon FIIs. In a range from 10-1 to 10-5 the melting curve was clearly visible and the melting temperature was stable. The melting temperature was identical when compared to the melting temperature observed for the cloned replicon. Further dilution of the DNA yielded a negative result. Comparison to agarose gel results showed that the intensity of the bands corresponded with the height of the melting curves (Figure 1). In addition,
the presence of more than one plasmid in one strain did not interfere with the accuracy and sensitivity of the melting curve assay (see Figure Ribonucleotide reductase 2). Figure 2 illustrates that the melting temperature of 84.6°C and 87.4°C from the two positive controls corresponded to the peaks visible in the tested strain. Figure 2 Detection of multiple WT plasmids shows the same melting curves as corresponding cloned replicon controls. The left panel
shows the melting curve of a WT strain with multiple plasmids. These plasmids were found to be of the ColE and F replicon. In the right panel the same result is obtained from two control strains each bearing either ColE or the F replicon. The melting temperature in the left panel peaks correspond exactly with the right panel peaks at 84.6°C and 86.4°C. Discussion The emergence of ESBLs has become an imminent threat to public health. This threat is emphasized by the continuous appearance of new β-lactamases. Although not all ESBL-enzymes pose the same threat, some facilitate a wide resistance to first-line antibiotics. To date, more than 900 different β-lactamases have been recognized . Of particular concern is the rapid spread of ESBLs, which is due to the location of the genes that encode them on transferable plasmids in Enterobacteriaceae. Identification of these replicons is useful for a better understanding of the epidemiology of the ESBL genes. For replicon identification Carattoli et al. developed a multiplex PCR-based replicon typing method .