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Selected Publication:

Type of publication: Doctoral Thesis
Type of document:

Year: 2013

Authors: Dzieciol, Monika

Title: Optimization of real-time PCR for detection and expression studies of fastidious microorganisms.

Other title: Die Optimierung der real-time PCR für den Nachweis von und für Expressionsstudien an anspruchsvollen Mikroorganismen

Source: Dissertation, Vet. Med. Univ. Wien, pp. 98.

Authors Vetmeduni Vienna:

Dzieciol Monika

Wagner Martin

Vetmed Research Units:
Institute of Food Safety, Food Technology and Veterinary Public Health, Unit of Food Microbiology

Graduation date: 04.03.13

Culture independent methods for identification of bacterial contaminants in the food chain are currently needed for food safety and public health. Especially for fastidious bacteria, which are capable of adapting to the environment, qPCR assays became effective tools in comparison to culture-dependent methods. The aim of the study was to develop and optimize qPCR assays for detection and quantification of the bacterial pathogens MAP, B. cereus and C. jejuni, with a particular usage of diagnosticians and expression studies. As a result, effective detection and quantification of bacteria from food matrices was possible. This provides an improvement for the respective food analytics and expression studies. For analyzing milk with a low MAP level, detection and identification by qPCR is the most valuable technique. The newly developed qPCR for detection of MAP presented in Publication I (DZIECIOL et al., 2010) is based on the MAP-specific Mptb52.16 target and was successfully applied on raw milk. The strategy used to optimize the limit of detection consisted of analyzing a maximum volume of sample in the qPCR. The results showed that concentrating the DNA of a large sample volume in combination with the qPCR assay permitted the quantification of low levels of MAP cells in raw milk (2.42×101 MAP cells/ml milk). Moreover, the assay provides an inherent possibility of differentiation between viable and dead MAP cells, offering better detection of metabolically active MAP in a sample. In Publication II (DZIECIOL et al., 2011), an RNA-based strategy was used to study the influence of bile salts on the multidrug CmeABC efflux pump and Cj0561c in poultry and humanC. jejuni strains. A newly designed and optimized RT-PCR method was successfully developed and allowed the description of the transcript levels of cmeABC and Cj0561c genes. Cj0561c gene was identified as strongly suppressed by the CmeR repressor, which also regulates the expression of CmeABC. The RT-PCR results suggested that, similar to cmeABC, Cj0561c could be induced by bile salts. In cultures without addition of bile salts, the transcript level of the Cj0561c gene was about tenfold higher than that of cmeABC. Multiplex qPCR for quantification and differentiation of emetic and non-emetic Bacillus cereusin milk products was successfully established, and described in Publication III (DZIECIOLet al., 2012). The assay based on the gyrase B (gyrB), the 16S rRNA, the ces and an IAC target resulted in an improved B. cereus group diagnostic tool. The limit of detection (1.91×103spores/ml) was improved by the application of mechanical disruption of spores combined with the milk protein lysis.

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