Rapid plasmid replicon typing by real time PCR melting curve analysis

M. Boot, S. Raadsen, P.H. Savelkoul, C. Vandenbroucke-Grauls

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)

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.
Original languageEnglish
Article number83
Number of pages5
JournalBMC Microbiology
Volume13
DOIs
Publication statusPublished - 15 Apr 2013

Keywords

  • ESBL
  • Plasmid
  • Replicon typing
  • SYBR-green
  • ESCHERICHIA-COLI STRAINS
  • BETA-LACTAMASE GENES
  • RESISTANCE
  • DISSEMINATION
  • CTX-M-15
  • BACTERIA
  • FI

Cite this

Boot, M. ; Raadsen, S. ; Savelkoul, P.H. ; Vandenbroucke-Grauls, C. / Rapid plasmid replicon typing by real time PCR melting curve analysis. In: BMC Microbiology. 2013 ; Vol. 13.
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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.",
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Rapid plasmid replicon typing by real time PCR melting curve analysis. / Boot, M.; Raadsen, S.; Savelkoul, P.H.; Vandenbroucke-Grauls, C.

In: BMC Microbiology, Vol. 13, 83, 15.04.2013.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - Boot, M.

AU - Raadsen, S.

AU - Savelkoul, P.H.

AU - Vandenbroucke-Grauls, C.

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N2 - 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.

AB - 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.

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KW - BETA-LACTAMASE GENES

KW - RESISTANCE

KW - DISSEMINATION

KW - CTX-M-15

KW - BACTERIA

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