No 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 (R 2 ) 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.

The Gram positive genus Listeria comprises six species, two of which -Listeria monocytogenes and Listeria ivanovii -are pathogenic. Both bacteria are facultative intracellular parasites able to infect macrophages and non-phagocytic cells, such as epithelial cells. After internalization, they undergo a characteristic intracellular infection cycle involving early escape from the phagocytic vacuole, rapid cytosolic replication, actin-based motility, and direct (cell-to-cell) spread to neighbouring cells, where the cycle begins again. Several virulence genes involved in key steps of this cycle are clustered together in a 9 kb locus that is located at the same chromosomal position in L. monocytogenes and L. ivanovii . This central virulence gene cluster or ' Listeria pathogenicity island 1 (LIPI-1)' is absent -or present in a non-functional formin the non-pathogenic Listeria spp. (Vázquez-Boland et al ., 2001a). LIPI-1 encodes a pore-forming toxin (listeriolysin O, LLO) and two phospholipases C (PlcA and PlcB) that cooperate to lyse the phagocytic vacuole membrane; an actin-polymerizing surface protein (ActA), responsible for intracellular bacterial motility and cell-tocell spread; a metalloprotease (Mpl) involved in the maturation of proPlcB; and a transcriptional activator (PrfA) that controls the expression of LIPI-1 genes and of other virulence determinants located elsewhere on the listerial chromosome (Portnoy et al ., 2002;Dussurget et al ., 2004). The latter include hpt , encoding a hexose phosphate transporter (Hpt) required for rapid cytosolic replication (Chico-Calero et al ., 2002), present in both L. monocytogenes and L. ivanovii ; and the inlAB operon, encoding two surface proteins (InlA and InlB) that mediate host cell invasion (Cossart et al ., 2003), only found to date in L. monocytogenes .