Development of immunological methods and Real-Time PCR for detection of Macadamia nut (Macadamia spp.)

Abstract: A new European labeling directive (2003/89/EC) states that certain foods and products derived thereof must always be declared. Among the tree nuts specified is Macadamia nut (Macadamia spp.). During the last few years, cases of IgE-allergic reactions, even severe anaphylaxes, have been reported. Reliable methods for the detection of this nut are needed. Protein from Macadamia nuts was isolated. Polyacrylamide gel electrophoresis in SDS revealed two main protein bands of about 20 and 50kDa. These protein bands were cut and extracted from the gel and rabbits were immunized with each protein. Immunoblotting showed dominant reactivity with the respective antigens. The antisera were further tested for specificity in immunodiffusion and in rocket immunoelectrophoresis. In addition, a specific DNA-method was developed, based on Real-Time PCR using Macadamia vicilin as target sequence. Two different primer pairs were tested. Specificity was tested against potentially related nuts. Optimisation of primer and probe concentrations was performed. The limit of detection was 2-4 pg DNA, corresponding to a macadamia nut concentration of 50 to 100 μg per g. In a background of soybean DNA, down to 0,01 % macadamia DNA could be detected.