A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control.
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.
Rodríguez-Lázaro
David
D
Veterinary Molecular Microbiology Section, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford BS40 5DU, United Kingdom. david.rodriguez@bris.ac.uk.
Pla
Maria
M
Scortti
Mariela
M
Monzó
Héctor J
HJ
Vázquez-Boland
José A
JA
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D017931:DNA Primers; D005516:Food Microbiology; D005784:Gene Amplification; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics*; D008461:Meat Products / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013552:Swine
D001427:Bacterial Toxins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes