title: 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.
authors.affiliation: Medical Microbiology & Infection Control, VU University medical center, De Boelelaan 1117, Postbus 70571007 MB, Amsterdam, The Netherlands.
authors.forename: Maikel
authors.initials: M
authors.lastname: Boot
authors.forename: Susanne
authors.initials: S
authors.lastname: Raadsen
authors.forename: Paul H M
authors.initials: PH
authors.lastname: Savelkoul
authors.forename: Christina
authors.initials: C
authors.lastname: Vandenbroucke-Grauls
mesh_terms: D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D004755:Enterobacteriaceae / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010957:Plasmids / Q000032:analysis* / Q000145:classification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature