Evaluation of locked nucleic acid and TaqMan probes for specific detection of cashew nut in processed food by real time PCR

Abstract

Cashew (Anacardium occidentale) nut can trigger serious reactions in allergic patients, including anaphylaxis and death. Labelling the presence of cashew nuts in food products is mandatory and consequently, sensitive and specific analytical methods must be developed. In this study, Ana o allergen coding sequences have been sequenced in several cashew varieties. Two hydrolysis probes, locked nucleic acid (LNA) and TaqMan, have been designed and their efficiency, sensitivity, limit of detection and specificity for Ana o 1 coding-sequence detection have been compared. Reliable Real Time PCR assays to detect and quantify up to 10 ppm of cashew nuts in complex mixtures have been developed. Moreover, the influence of boiling and autoclave treatment on cashew nut detectability has been analysed by qPCR, showing both probes similar performance. This analytical method was able to detect up to 1000 ppm with good functionality in autoclave treated samples. Boiling did not affect cashew nut detectability. Both hydrolysis probes are suitable for Ana o 1 coding sequence detection. Applicability of the assay has been studied by analysing several food products, and comparing the results with those of a commercial ELISA kit.