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. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method.

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. This method was successful for both intended uses: (1) direct detection and quantification of Y. enterocolitica in artificially and naturally contaminated raw milk samples and (2) characterization of growth potential of different serotypes of Y. enterocolitica in raw milk at the most commonly used storage temperatures. The recent method eliminates the pre-PCR enrichment step, which makes it possible to quickly assess milk-related consumer exposure to this pathogen.

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. Analysis of competition between the individual reactions within the multiplex real-time PCR assay showed that GI and GII NoV plasmid DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively, while a 4 log excess between GI and GII plasmid DNAs hindered amplification of the target with the lowest concentration. High concentrations of the real-time PCR IAC (MNV-1 plasmid DNA) also interfered with the possibility of the developed multiplex real-time RT-PCR assay to detect quantitatively and simultaneously the presence of GI and GII NoVs within one sample. The specificity of the multiplex assay was evaluated by testing a NoV RNA reference panel containing nine GI, eight GII, and one GIV in vitro synthesized RNA fragment, plus 16 clinical samples found positive for GI and GII NoVs previously. In addition, a collection of bovine NoVs and other (non-NoV) enteric viruses were found to be negative, and no cross-amplification between genogroups was observed.

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. Also the effect of sublethal acid stress exposure on the expression levels of several virulence genes and on the capacity to invade Caco-2 cells was analyzed. Acid-adapted and non-adapted cells showed different acid tolerance response (ATR) patterns. Our data also demonstrate that pre-exposure to sublethal acid stress might lead to gadD2 induction. However, no correlation with the origin of the strain and with the ATR was noticed, indicating that probably other genes also could play an important role in this ATR mechanism. Additionally, our data showed that acid adaptation could influence the virulence capacity by regulating the expression levels of virulence genes. Moreover, the inlA expression data strongly correlated with the results of the in vitro invasion study. These results lead to the indication that low pH and acetic acid, used in minimally processed food products, might influence the virulence potential of L. monocytogenes.

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. Results of this experiment indicated that evaporation of water occurred during real-time PCR runs regardless of the DNA type, the seal type and whether or not 7 microl of mineral oil was used as cover layer. Data from this experiment also suggested co-evaporation of DNA, with an apparent negative correlation between the size of the DNA type and the extent of this co-evaporation. The use of 7 microl of mineral oil as cover layer seemed to prevent to some extent co-evaporation of DNA. The use of plasmids as standard combined with 7 microl of mineral oil as cover layer in the real-time PCR setup resulted in a complete absence of positive NTCs while only minor effects were noticed on the performance of the real-time PCR. In general, our results showed that the high sensitivity of an optimized real-time PCR assay should be considered as--besides a great advantage--a potential risk factor for obtaining false-positive results when using this technique.

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 (

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. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.

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. The expression of mntH and the different control genes (16S rRNA, rpoD and gmk) varied according to the growth phase. For mntH a maximum in the expression was detected in the early exponential phase. To obtain an accurate quantification and reliable comparison of the mntH expression in different S. enterica strains under various biological conditions, the ratio mntH mRNA level to the normalization factor was determined. The latter is the geometric mean of the RNA level of three housekeeping genes 16S rRNA, rpoD and gmk calculated by the geNorm program. MntH was basally expressed in all tested S. enterica strains and induced by hydrogen peroxide (H(2)O(2)) or ethylenediaminetetraacetic acid (EDTA) in Brain Heart Infusion. Under the nutrient limiting conditions of Sauton medium, the basal mntH expression was higher than in BHI, whereas H(2)O(2) induced the expression 40 times. A similar induction was obtained for Salmonella in porcine peripheral blood monocytes (PBM).

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. monocytogenes was detected in 23.4% of the samples. The highest prevalence of L. monocytogenes was found in prepared minced meat (42.1%) and smoked halibut (33.3%). Contamination levels were in most cases low (<10 CFU/g); however, levels higher than 100 CFU/g were detected in some samples of smoked salmon, smoked halibut, and prepared minced meat. A high prevalence of Listeria innocua (15.8%) and Listeria welshimeri (36.8%) was detected in prepared minced meat. L. monocytogenes strains isolated from different contaminated products were subjected to repetitive element sequence-based PCR (REP-PCR) typing to determine possible associations with product type, producer, or market. REP-PCR patterns were analyzed using BioNumerics software, and seven different groups with at least 90% similarity were identified. The cluster analysis indicates that cross-contamination occurred at the producer and retail level. Serotype identification of the strains by PCR revealed that most belonged to the 1/2a(3a) serotype group.