Rapid plasmid replicon typing by real time PCR melting curve analysis

Abstract

Background: Genes encoding Extended Spectrum Beta Lactamases are usually located on transferable plasmids. Each plasmid contains its own replication mechanism. Carattoli et al. developed an extended PCR-based replicon typing method to characterize and identify the replicons of the major plasmid incompatibility groups in Enterobacteriaceae. Based on this method, we designed a rapid approach with amplicon detection by real-time melting curve analysis. This method appeared to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment.

Results: We successfully integrated the post-PCR analysis of the replicon typing into a closed system, which leads to a 10-fold increase in sensitivity compared to agarose gel visualization. Moreover, the use of crude lysates and SYBR-green saves a considerable amount of hands-on time without decreasing the sensitivity or specificity.

Conclusions: This real-time melting curve replicon typing method appears to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment.

Figure 1

Melting curves of serial dilutions of the FIIs replicon. The melting curves intensity differences based on 10^-1 to 10^-9 dilutions of the FIIs replicon (melting peak at 87.4 average for this experiment). For each melting curve the corresponding agarose band is presented in the grey box. Shown in pairs are the curves that gave a positive result both as melting curve and after visualization on agarose gel (blue = 10^-1, purple = 10^-2, green = 10^-3, red = 10^-4 and turquoise = 10^-5). The dilutions from 10^-6 to 10^-8 are visible as peaks of 4300 y (10^-6) to 4117 y (10^-8). These are not shown as agarose bands because they were not visible on agarose gel.

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.