A qPCR assay to detect and quantify Shiga toxin-producing E. coli (STEC) in cattle and on farms: a potential predictive tool for STEC culture-positive farms.

Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle.

Quantification of Yersinia enterocolitica in raw milk using qPCR.

This report describes a new, sensitive and specific protocol for rapid detection and quantification of Yersinia enterocolitica in artificially contaminated raw milk samples. The new method is based on an optimized real-time PCR protocol with a TaqMan probe. The primers and probe are based on the chromosomal ail gene.

Multiplex real-time RT-PCR for simultaneous detection of GI/GII noroviruses and murine norovirus 1.

A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR detection assay described recently was integrated successfully into the multiplex assay, making it possible to detect GI and GII NoVs and MNV-1 in one reaction tube with MNV-1 plasmid DNA as real-time PCR internal amplification control (IAC). The results showed a nearly complete concordance between the multiplex assay and the corresponding single-target PCRs.

Influence of acid stress on survival, expression of virulence genes and invasion capacity into Caco-2 cells of Listeria monocytogenes strains of different origins.

The influence of food-related acid stress on the virulence capacity of Listeria monocytogenes was evaluated. The survival of acid-adapted and non-adapted L. monocytogenes cells during exposure to lethal concentrations of acetic acid was monitored.

Laboratory efforts to eliminate contamination problems in the real-time RT-PCR detection of noroviruses.

In the current study, laboratory efforts to prevent the presence of positive NTCs (no template controls) during the optimization of a quantitative real-time reverse transcriptase PCR assay for detection of Noroviruses (NoVs) are described. Two DNA types (single-stranded (ss)DNA fragments and plasmid DNA) were used to generate a real-time PCR standard and a high frequency of positive NTCs was noticed in the case of ssDNA fragments. To investigate our suspicion of well-to-well migration of DNA during real-time PCR runs as possible cause of the positive NTCs, an "evaporation-experiment" was set up in which the evaporation of water and the possible co-evaporation of DNA were measured as a function of the DNA type (ssDNA-fragments, plasmid DNA and genomic DNA), the reaction plate seal type (adhesive film or 8-cap strips) and the use of 7 microl of mineral oil as cover layer.

Quantification of gene expression of Listeria monocytogenes by real-time reverse transcription PCR: optimization, evaluation and pitfalls.

In the current study, various steps in the real-time reverse transcription PCR (real-time RT-PCR) method for determination of RNA expression levels starting from different numbers of Listeria monocytogenes cells were evaluated and optimized. Our results showed that the RNA isolation method as well as the cDNA synthesis may influence the sensitivity of the procedure. For high bacterial cell numbers (10(9) bacterial cells), the RNAqueous kit and the RNeasy Mini kit were equally useful, whereas for low bacterial cell numbers ( View Article and Find Full Text PDF

Differential inlA and inlB expression and interaction with human intestinal and liver cells by Listeria monocytogenes strains of different origins.

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed.

Real-time reverse transcription PCR for the quantification of the mntH expression of Salmonella enterica as a function of growth phase and phagosome-like conditions.

This article presents an experimental design for measuring the mRNA expression in Salmonella enterica of the mntH gene in phagosome-mimicking conditions. The expression of mntH was quantified by real-time reverse transcription PCR for different S. enterica strains of porcine origin under different biological growth conditions which mimicked the environment inside the phagosome.

Improved detection of Mycobacterium paratuberculosis in milk.

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.

Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market.

Listeria monocytogenes is a major concern to producers of ready-to-eat foods because of the high mortality rate associated with listeriosis and the widespread nature of the organism. To investigate the prevalence of this pathogen in different ready-to-eat food products on the Belgian market, a variety of 252 ready-to-eat food products, mainly fish and meat products, were analyzed. Overall, L.