Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Detection of Peanut Allergen by Real Time PCR: Looking For the Suitable Detection Marker and Dna Isolation Method

Version 1 : Received: 13 April 2021 / Approved: 14 April 2021 / Online: 14 April 2021 (14:10:52 CEST)

How to cite: Sanchiz, A.; Sánchez-Enciso, P.; Cuadrado, C.; Linacero, R. Detection of Peanut Allergen by Real Time PCR: Looking For the Suitable Detection Marker and Dna Isolation Method. Preprints 2021, 2021040382 (doi: 10.20944/preprints202104.0382.v1). Sanchiz, A.; Sánchez-Enciso, P.; Cuadrado, C.; Linacero, R. Detection of Peanut Allergen by Real Time PCR: Looking For the Suitable Detection Marker and Dna Isolation Method. Preprints 2021, 2021040382 (doi: 10.20944/preprints202104.0382.v1).

Abstract

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitive population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergenic population. The development of suitable analytical methodologies to detect this allergen in processed foods is advisable. Real Time PCR allowed a specific and accurate amplification of allergen sequences. The optimal genome targets for specific and sensitive detection are those that show interspecific variation and high copy number. Some food processing methods could induce structural and/or conformational changes in proteins and produce the fragmentation and/or degradation of genomic DNA. In this work, we use chloroplast markers for specifically detection of peanut by Real Time PCR, in order to increase the sensitivity. Three different protocols of DNA isolation were evaluated, for total and organelle-DNA extraction. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100000 to 0.1 ppm. DNA isolation from peanut, mixtures and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA was evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16 and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Moreover, the influence of pressure and thermal processing on the peanut detectability was analyzed.

Subject Areas

Real Time PCR; peanut; food allergen; chloroplast marker; DNA isolation

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