Molekularbiologischer Nachweis von Enterobacter sakazakii (Cronobacter spp.) in Säuglings-und Kleinkindernahrung
Beschreibung
vor 15 Jahren
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.
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.
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