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Funk, Anne Edith (2010): Molekularbiologischer Nachweis von Enterobacter sakazakii (Cronobacter spp.) in Säuglings-und Kleinkindernahrung. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Detection of Enterobacter sakazakii (Cronobacter spp.) in powdered infant formulae using a molecular biological method. The aim of the studies presented here was the development of a screening test based on PCR technique for the detection of Enterobacter (E.) sakazakii (Cronobacter spp., IVERSEN et al., 2008a) in powdered infant formulae. Variable regions of the 16S RNA gene of E. sakazakii were chosen for amplification by primers designed with the help of the computer programme Primer3 (ROZEN und SKALETSKY, 2000). Chimeric primer were used as internal amplification control. To check whether the amplicon was generated agarose gel electrophoresis was employed. To test the sensitivity and specificity of the PCR method 57 E. sakazakii strains as well as 169 isolates belonging to 16 species or genera of other bacteria than E. sakazakii were used. Before testing, the E. sakazakii strains were examined by RAPD-PCR to ensure individuality. The PCR method gave positive results for all 57 E. sakazakii strains, three Hafnia alvei isolates, however, were also positive. It could be shown by DNA sequence analysis that H. alvei has a16S rRNA gene sequence identical with the target DNA used for the detection of E. sakazakii. Therefore, the amplicons of the E. sakazakii strains and the H. alvei isolates were analysed by restriction digest using MvaI as restriction enzyme. Both species could be differentiated clearly by their electrophoretic band patterns. To test the performance of the PCR method if food has to be examined 52 samples of powdered infant formulae were artificially contaminated with ten different strains of E. sakazakii (5 to 24 bacteria per 100 g). The samples were from four producers and they included different types of infant formulae, follow-up formulae and powdered formulae for special medical purposes. 100 g in each case of the artifically contaminated as well as 100 g in each case of non contaminated (negative control) sample material was preenriched in buffered peptone water for 24 h at 37 °C. After preenrichment DNA was extracted using a commercial test kit (PrepMan Ultra) and tested by the PCR method. A culture technique for the detection of E. sakazakii (FDA/SCAN, 2002) was carried along as reference method. E. sakazakii was detected in all samples with both methods.