Detection of wheat as an allergenic substance in food by a nested PCR method

Abstract

A nested PCR method was developed for the detection of DNAs extracted from allergenic substances (here, wheat) in food. Because of DNA fragmentation, detection of wheat-specific DNA extracted from food, such as retort pouch food, is very difficult. Therefore, to improve the sensitivity of detection, a nested PCR primer pair (Wtr01NE2-5′ and Wtr10NE5-3′: amplicon size 97 bp) was newly designed within the region of the PCR products amplified by the official Japanese primer pair (Wtr01-5′ and Wtr10-3′; amplicon size 141 bp) for wheat. Genomic DNAs of seven kinds of commercial processed foods containing wheat, wheat flour and three kinds of wheat flours pressure-heated at 100, 121 and 131°C were extracted with a commercial ionexchange type kit by modifying the Japanese official method. The nested PCR method involved two PCR procedures. First, PCR was performed by varying both the PCR reagents and cycling conditions of the Japanese official method. Second, PCR was performed using the first PCR products diluted 200-fold with TE buffer. The Japanese official method enabled detection of only four of the seven kinds of foods and three of the four kinds of flours (one sample was just a trace), while the nested PCR method detected all seven foods and all four flours. Investigation of the detectability of the four kinds of wheat flours depending on the size of the amplified fragment using five primer pairs showed that its size must be kept to less than approximately 100 bp. The nested PCR method significantly improved the sensitivity of detection of wheat-specific DNA.