Detailansicht

Comparison of two ELISAs and one real-time PCR method for the detection of potentially allergenic sesame in food

Ines Elisabeth Bretbacher

Art der Arbeit

Diplomarbeit

Universität

Universität Wien

Fakultät

Fakultät für Chemie

Betreuer*in

Margit Cichna-Markl

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DOI

10.25365/thesis.5406

URN

urn:nbn:at:at-ubw:1-29975.57306.436461-7

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(Print-Exemplar eventuell in Bibliothek verfügbar)

Abstracts

Abstract

(Deutsch)

Vergleich von einem Sandwich und einem kompetitivem ELISA und einer real-time PCR Methode zur Detektion von potentiell allergenem Sesam in Lebensmitteln Lebensmittelallergien treten in unserer Gesellschaft immer häufiger auf. Bereits 6-8\% der Kinder leiden an einer Lebensmittelallergie. Symptome reichen von leichten Beschwerden wie Juckreiz oder Schnupfen bis hin zu Verdauungsbeschwerden und schlimmstenfalls zum anaphylaktischen Schock. Die einzige Möglichkeit eine allergische Reaktion zu vermeiden, ist der strikte Verzicht auf das jeweilige Lebensmittel. 14 Nahrungsmittelzutaten, die besonders häufig Allergien auslösen, müssen auf der Verpackung deklariert werden (EU-Richlinie (2007/68/EG). Auch Sesam gehört zu dieser Gruppe, denn bereits kleinste Spuren können eine allergische Reaktion auslösen. Es wurden bereits einige Methoden zur Detektion von allergenen Lebensmitteln entwickelt. ELISAs und Polymerase Kettenreaktion sind bislang die Methoden der Wahl. Beide weisen einige Vor- sowie auch einige Nachteile auf. Es gibt jedoch kaum Studien, in denen die Methoden direkt miteinander verglichen wurden. Ziel dieser Arbeit war es, einen Sandwich und einen kompetitiven ELISA, und eine real-time PCR Methode zur Detektion von Spuren von Sesam in Lebensmitteln zu vergleichen. Um die Methoden zu vergleichen, wurden 4 Lebensmittelmatrizes (Vollkornkeks, Vollkornbrot, Zwieback und Müsli) mit gemahlenen Sesamkörnern in verschiedenen Konzentrationen (0.001\% - 1\%) gespikt. Die Proteine wurden mit einem Extraktionspuffer, DNA mittels der CTAB-Methode oder dem QIAmp Stool Kit extrahiert. Während es bei der Proteinextraktion keine Schwierigkeiten gab, bereitete die Extraktion der DNA aus Vollkornbrot größere Probleme. Im Sandwich ELISA wurde bei einer 1:1 Verdünnung des Extrakts eine Nachweisgrenze (S/N=3) von 4 ppm und im kompetitiven ELISA von 9 ppm erreicht. Mit der PCR Methode wurde in Vollkornkeks und Zwieback eine Nachweisgrenze von 50 ppm erreicht. In Vollkornbrot betrug die Nachweisgrenze 500 ppm und in Müsli nur 5000 ppm.

Abstract

(Englisch)

Comparison of two ELISAs and one real-time PCR method for the detection of potentially allergenic sesame in food Sesame is one of the allergenic foods which has to be labeled in the food ingredient list according to EU regulations. Recently, in our research group three analytical methods have been developed allowing the detection of traces of sesame in food. These methods include a sandwich ELISA, a competitive ELISA and a real-time PCR method. The aim of the diploma thesis was to spike different food matrices with known amounts of sesame and to analyse the spiked samples with each of the three methods to compare their applicability and performance. Four food matrices (rusk, whole wheat cookies, whole wheat bread and muesli) were spiked with sesame in concentrations from 0.001% to 1%. The protein fraction of the food samples were extracted with a protein extraction buffer containing Tris and NaCl. DNA was extracted either with the CTAB method or with a commercial DNA extraction kit. When the protein extract was diluted 1:20 (1 mL + 19 mL), the limit of detection of the sandwich ELISA was found to be 44 ppm. Loading the protein extract diluted 1:1 (1 mL + 1 mL) resulted in a LOD of 4 ppm. The limit of detection of the competitive ELISA was higher. Diluting the food extracts 1:20 resulted in a LOD of 90 ppm, by using the extract diluted 1:1 a LOD of 9 ppm was achieved. For whole wheat cookies the recoveries obtained with the sandwich ELISA were in the range from 80 to 100%. Only at a sesame concentration of 0.1% sesame, the recovery of sesame was 200%. With the competitive ELISA, recoveries >100% were obtained at lower spiking levels (0.05, 0.005 and 0.001%). With the real-time PCR method, sesame could be detected down to a spike level of 0.005%. The amplification efficiency in whole wheat cookies was 81.4%. For whole wheat bread, in general too high recoveries (about 200%) were obtained with the sandwich ELISA. In contrast, with the competitive ELISA recoveries of 100% were achieved for the 0.005 and 0.001% spike level. However, for the other spike levels the recovery was less than 100%. Extracting a high amount of pure DNA from whole wheat bread with the CTAB method proved to be difficult. Low amounts of DNA were also obtained with the QIAmp Stool Kit. The LOD of the PCR method in whole wheat bread was determined to be 0.05% sesame. In the case of rusk, for all spike levels recoveries > 100% were obtained with the sandwich ELISA. With the competitive ELISA, at lower spike levels even recoveries up to 300% were obtained. DNA extraction with the CTAB method yielded DNA of sufficient amount and purity. Sesame DNA could be amplified down to a sesame concentration of 0,005%, the amplification efficiency was 76%. The analysis of sesame spiked muesli with the sandwich ELISA resulted in too high recoveries. In contrast, at higher spike levels recoveries of 100% were obtained with the competitive ELISA, only at a spike level of 0.001% the recovery was 300%. DNA extraction with the CTAB method yielded high DNA concentration and purity. Nevertheless, sesame DNA could not be amplified in any of the spiked samples. Inhibition control tests indicated that the results were not caused by enzyme inhibition. When DNA was extracted with the QIAmp Stool Kit, sesame DNA could be amplified down to a sesame concentration of 0.5% sesame. The ELISA methods proved to be better applicable to the detection of traces of sesame in food. The LOD of both ELISA methods was found to be lower than the LOD of the PCR method. In addition, even from complex food matrices proteins could be extracted without any problems. Compared to the competitive ELISA, the advantages of the sandwich ELISA are the absence of matrix effects and its lower limit of detection.

Autor*innen

Ines Elisabeth Bretbacher

Haupttitel (Englisch)

Comparison of two ELISAs and one real-time PCR method for the detection of potentially allergenic sesame in food

Paralleltitel (Deutsch)

Vergleich von einem Sandwich und einem kompetitivem ELISA und einer real-time PCR Methode zur Detektion von potentiell allergenem Sesam in Lebensmitteln

Publikationsjahr

2009

Umfangsangabe

109 S. : graph. Darst.

Sprache

Englisch

Beurteiler*in

Margit Cichna-Markl

Klassifikation

35 Chemie > 35.23 Analytische Chemie: Allgemeines

AC Nummer

AC08101861

Utheses ID

4838

Studienkennzahl

UA | 190 | 423 | 477 |

Detailansicht

Abstracts

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