Diagnostic PCR for the Detection of Yersinia enterocolitica and Salmonella in the Food-Chain: Reliability of PCR Performance
Abstract: This thesis deals with the methodological advances of diagnostic PCR including reliability of PCR and pre-PCR processing of food and feed samples. Diagnostic PCR has been greatly improved by the introduction of the second generation of PCR, so-called real-time PCR. Automated closed-tube quantitative real-time PCR favours the analysis of food-borne pathogenic bacteria. However, in common with conventional PCR, the real-time PCR technology requires pre-PCR processing of the food/feed sample (i) to remove PCR-inhibitory substances, (ii) facilitate detection of low concentrations of target bacteria, (iii) to convert a heterogeneous bulk sample to a more homogeneous PCR sample, and (iv) to restrict competitive background flora. The aim of the research presented in this thesis was to adapt microbiological food sampling to PCR by designing pre-PCR processing strategies for future routine analysis of Yersinia enterocolitica and Salmonella in complex samples from the food-chain. To enable the detection of low concentrations of Y. enterocolitica and Salmonella in PCR-inhibitory samples, such as pork and animal feed, enrichment PCR procedures have been employed. The reliability of PCR detection of pathogenic Y. enterocolitica using a developed multiplex PCR assay was studied using a logistic regression model for determination of the detection probability. The probability of detecting 1´104 CFU/ml Y. enterocolitica was estimated to be 85.4%. A Yersinia-PCR-compatible enrichment (YPCE) medium was developed to remove the necessity for sample preparation prior to PCR detection of Y. enterocolitica. The pre-PCR processing strategy allows detection of low concentrations (101 CFU/ml) in the presence of background flora in concentrations up to three orders of magnitude higher than Y. enterocolitica. The YPCE medium can, form part of integrated and automated pre-PCR processing protocol, especially for swab samples. To complement the Yersinia assay a RAPD protocol was developed for inter-laboratory use. The stringent RAPD protocol was evaluated on 70 Yersinia strains and allowed discrimination at serotype level, and the sub-clusters of Y. enterocolitica correlated with the geographic origin of isolates, where especially O:3 strains from Scandinavia formed a homogeneous sub-cluster. Enrichment PCR of Salmonella enterica was studied using real-time PCR. A model was developed to describe the 5' nuclease real-time PCR performance in the presence buffered peptone water (BPW) and brain heart infusion. Using the model it was found that the rTth DNA polymerase mixture was more resistant to the presence of BPW than AmpliTaq Gold. Accurate detection of 1 CFU/ml S. Enteritidis inoculated in BPW required 8.4 hours’ enrichment using the rTth DNA polymerase mixture, while AmpliTaq Gold required 11.6 h. Using an alternative DNA polymerase, Tth instead of Taq, facilitated the PCR detection of Salmonella in animal feed. The PCR protocol was more sensitive than the traditional culture-based standard method (NMKL-71), since out of 155 feed samples, 8% were positive for PCR detection of Salmonella in comparison with 3% with the NMKL-71 method.
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