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 language | English |
---|---|
Pages (from-to) | 9008-12 |
Number of pages | 5 |
Journal | Applied and Environmental Microbiology |
Volume | 71 |
Issue number | 12 |
Publication status | Published - Dec 2005 |
Keywords / Materials (for Non-textual outputs)
- 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