A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control

David Rodríguez-Lázaro, Maria Pla, Mariela Scortti, Hector J Monzó, Jose A Vazquez-Boland

Research output: Contribution to journalArticlepeer-review

Abstract

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.
Original languageEnglish
Pages (from-to)9008-12
Number of pages5
JournalApplied and Environmental Microbiology
Volume71
Issue number12
Publication statusPublished - Dec 2005

Keywords

  • Animals
  • Bacterial Toxins/genetics
  • DNA Primers
  • Food Microbiology
  • Gene Amplification
  • Heat-Shock Proteins/genetics
  • Hemolysin Proteins
  • Listeria monocytogenes/genetics
  • Meat Products/microbiology
  • Polymerase Chain Reaction/methods
  • Swine

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