Abstract: A multiplex ligation-dependent probe amplification (MLPA) technique was developed for simultaneous detection of six food allergens: soy, sesame, peanut, almond, hazelnut and walnut. Six pairs of species-specific MLPA probes targeting the internal transcribed spacer (ITS) sequences were designed and synthesized. The probes were hybridized with the target DNA, ligated and amplified by polymerase chain reaction (PCR) and the amplicons were detected using capillary electrophoresis, revealing that they could clearly discriminate the six food allergens. Good specificity of the MLPA system was observed for more than 20 plants, animals and bacteria. The limit of detection (LOD) of the assay was determined to be 5 mg/kg for model cookie spiked with different concentrations of the target species. The applicability of the MLPA assay was demonstrated through the analysis of 30 different commercial products, evidencing good performance in practical applications. Therefore, the MLPA technique is highly specific and sensitive and can provide technical support for the assessment, label management and risk control of food allergens.

Key words: multiplex ligation-dependent probe amplification; food allergen; multiple detection; limit of detection