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Open Access Highly Accessed Methodology article

Rapid plasmid replicon typing by real time PCR melting curve analysis

Maikel Boot, Susanne Raadsen, Paul HM Savelkoul and Christina Vandenbroucke-Grauls*

Author Affiliations

Medical Microbiology & Infection Control, VU University medical center, De Boelelaan 1117, Postbus 70571007 MB, Amsterdam, The Netherlands

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BMC Microbiology 2013, 13:83  doi:10.1186/1471-2180-13-83

Published: 15 April 2013

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.

Keywords:
ESBL; Plasmid; Replicon typing; SYBR-green