Detection of Lupine (Lupinus spp. L.) as a food allergen using three methods: end-point PCR, Real-Time PCR and Elisa
Keywords:lupine, food allergy, end-point PCR, real-time PCR, ELISA
The aim of this work was to compare three methods for the detection and quantification of lupine as an allergen in food. The methods that were used in this work were the direct method: ELISA and the indirect methods: end-point PCR and real-time PCR. We examined the detection limit (the sensitivity with which we can detect the presence of the allergen in a sample) and the reliability for performing an analysis. We used 17 samples of plant species from a processing plant for dehydrated soups production and lupine samples from lupine processing companies. Its practical use is wide and it is used mainly in the bakery industry, in the manufacture of confectionery, pasta, sauces, as a substitute for soy and also in the production of gluten-free food, because it does not contain gluten. Lupine, however, is also included in the list of 14 allergenic substances, which in accordance with the EU legislation must be listed on food labels. The high risk group, which suffers from primary sensitization or cross-reaction with peanuts, are allergic patients. In the EU, people who are allergic to peanuts range from 0.7 to 1.5%. In experiment 1, we detected the presence of lupine using primers for the detection of α- and δ-conglutine in the samples, using the end-point PCR method and the detection limit of this reaction was at the level of 100 ppm. For the vizualization of the DNA fragments, we used a 2% agarose gel and UV visualizer. In experiment 2 we detected lupine using the TaqMan real-time PCR reaction and primers for the detection of α and δ-conglutine at the level of 10 ppm of lupine in sample. The CP values of lupine using primers for the detection of α-conglutine was 24.85 ± 0.12 and the reliability equation was R2 = 0.9767. The CP lupine values using primers for the detection of δ-conglutine was 22.52 ± 0.17 and the reliability equation was R2 = 0.9925. In experiment 3, we used a sandwich ELISA method for the detection of lupine and the detection limit was within the range of 2-30 ppm and the reliability of the method according to the reliability equation was R2 = 0.9975. The high sensitivity and equation of reliability justify the use of these methods for the detection and quantification of lupine in practice. The most sensitive indirect method for the detection of lupine in our study was the method of real-time TaqMan PCR with a detection limit 10 000-10 ppm of lupine. The most sensitive direct methot was ELISA with detection limit 2-30 ppm of lupine.
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