Evaluation and optimization of four real-time PCRs, using TaqMan-probes, for detection of and discrimination between barley, oat, rye and wheat

• Main Author: Björklund, Kristofer
• Format: Others
• Language: English
• Published: Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi 2008
• Subjects: Gluten containing cereals; coeliac disease; cereal-specific analysis; gluten-free food; polymerase chain reaction; Medical laboratory science; Medicinsk laboratorievetenskap
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9171

Coeliac disease is a chronic inflammatory disease treated with a gluten-free diet, excluding barley, rye and wheat. Hence, there is a demand for methods able to detect gluten in foods in order to ensure correct labeling of products. According to the Codex Alimentarius Commission, 20ppm gluten is the maximum amount allowed in food labeled gluten-free. PCR can detect DNA from cereals in food. Four real-time PCR-systems, using TaqMan®-probes for detection of barley, oat, rye and wheat were optimized and evaluated. Evaluations were carried out using seeds. Primers were targeted to genes coding for prolamines, seed storage proteins. PCR-systems targeted to barley, oat and wheat were shown to be specific for the cereals corresponding to each system. The system targeted to rye showed cross-reactions with durum wheat and spelt wheat. Detection limits were 50pg, corresponding to <10 haploid genome copies for each cereal. All systems were able to detect 250ppm amounts of DNA, most likely even smaller amounts are detectable. All systems showed an amplification efficiency of ≥95%. Systems for detection of barley, oat and wheat are ready for further evaluation, using food products as samples. The rye system however, needs to be re-designed before further evaluation can take place.