Real-time PCR Detection Methods for Food Allergens (Wheat, Buckwheat, and Peanuts) Using Reference Plasmids.

Specific and sensitive real-time qualitative polymerase chain reaction (PCR) methods for the detection of food allergens including wheat, buckwheat, and peanuts were developed that could cancel between instrument effects and avoid risks of false-positives and false-negatives. In these real-time PCR analysis, the cutoff for determination of positive samples was set in every PCR run by using reference plasmids containing known copies of the target sequences. The copy numbers of the plasmids were used to detect the allergenic ingredients corresponding to 10 ppm (w/w) protein in highly processed foods (cooked for more than 30 min at 122 °C). Reference plasmid analysis for each real-time PCR run helped to minimize variability between runs and instruments (7900HT Real-Time PCR systems and Light Cycler Nano). It also helped to avoid false positives due to trace levels of contaminants from the laboratory environment or agricultural products. The specificity of the real-time PCR method was verified using 79 commonly used food materials and some of their relatives. The method was sensitive enough to detect those allergenic ingredients corresponding to 10 ppm (w/w) in seven types of incurred samples. The current official Japanese method was not able to detect the allergens in some of the incurred samples. The developed method can avoid false negatives due to lack of sensitivity and is useful to confirm positive ELISA screening tests.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation.

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

[Applicability of PCR methods for detection of shrimp and crab in processed food].

According to Japanese food allergen labeling regulations, an ELISA screening test is used for detection of crustacean proteins in food and a shrimp/crab-PCR confirmation test is used to confirm a positive ELISA screening test and to exclude false positives. Forty-six kinds of processed foods labeled as containing shrimp/crab were subjected to ELISA screening test and PCR confirmation test and the usefulness of the shrimp/crab-PCR was evaluated. Twenty-seven of the 46 samples contained total crustacean protein levels of 10 ppm or more in the ELISA screening test. All of the samples were positive in the shrimp/crab-PCR confirmation test. The results of the confirmation test were consistent with the declaration in the list of ingredients and with the results of the ELISA screening test. The shrimp/crab-PCR confirmation test was demonstrated to be applicable to various kinds of foods, including powder, extract, seasoning paste, prepared frozen food, snack food, retort food and canned food.