Entwicklung und Anwendung enzymimmunologischer Verfahren zum Nachweis, zur Speziesdifferenzierung und zur Serotypisierung von Cronobacter sakazakii
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Beschreibung
The present work deals with the development and application of enzyme-immunological method for the detection and characterization of Cronobacter sakazakii in dried infant formulas.
In order to obtain polyclonal antibody, two immunizations were carried out in rabbits. The immunreagent was made up of three different Cronobacter sakazakii strains which were inactivated by paraformaldehyde (PFA). For the first immunization, a mixture of three different strains serotypes (O1, O3, O7) was used. The second immunization consisted of a mixture of strains of serotype O2. The immune response was studied using a direct enzyme immunoassay.
The obtained antisera were precipitated by means of ammonium sulfate and a share then biotinylated. Using the precipitated antiserum as capture antibodies and the precipitated and biotinylated antiserum as detection antibodies two sandwich enzyme immunoassay were developed (Cronobacter1 and Cronobacter2).
To determine the testspecifity 296 different Cronobacter sakazakii strains have been investigated in conditions of the enzyme immunoassays. Detectability and cross-reactions with other Cronobacter spp. and related bacterial strains were tested. Both sandwich EIA gathered 73% of Cronobacter sakazakii strains together, there were no cross-reactions with other Cronobacter spp. or related bacteria found. The detection limits for isolates of the different serotypes varied between 103-105 cfu / ml. After standard enrichment in buffered peptone water at 37 ° C for 18 +/- 2 hours and use of undiluted broth in EIA, the positive strains obtained always distinct color reactions. The implementation of the detection method in our experiments took 21 to a maximum of 24 hours, including the pre-enrichment.
There were 79 strains of Cronobacter sakazakii serotyped by multiplex PCR according to the method of Sun et al. (2012). The results were compared with the results of the sandwich EIA. The sandwich EIA Cronobacter1 demonstrated specific detection of all strains of serotype O1. In addition, it showed color reactions with about half of the strains of serotypes O3 and O7. The reference curves showed significantly different dose-response relationships for the strains of different serotypes. The test system Cronobacter2 reacted highly specific with all strains of serotype O2. Both test systems showed no cross-reactivity with Cronobacter sakazakii strains of other serotypes.
To check the influence of the sample matrix on the test systems Real samples dried infant formulas were artificially contaminated. Following the investigation transition to ISO / TS 22964: 2006, the samples were dissolved in sterile, buffered peptone water, artificially contaminated with 1-10 cfu different Cronobacter sakazakii strains and incubated. The sample matrix showed no influence on the test systems. The results of the culturally-microbiological reference methods and the developed enzyme immunoassay systems showed a match in 100% of the audited sample.
Autor*in
Mara Benecke
Themen in »Entwicklung und Anwendung enzymimmunologischer Verfahren zum Nachweis, zur Speziesdifferenzierung und zur Serotypisierung von Cronobacter sakazakii«
Cronobacter sakazakii Säuglingsanfangsnahrung Enzymimmunoassay (EIA) Sandwich-Enzymimmuntest Serotypisierung Lebensmittelmikrobiologie