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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.

SUBMITTER: Boot M

PROVIDER: S-EPMC3639092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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