A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods

Abstract

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.

The Development of Highly Specific and Sensitive Primers for the Detection of Potentially Allergenic Soybean (Glycine max) Using Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD)

“…Thus, even low levels of soybean DNA resulted in a clearly visible positive test line. By contrast, protein-based LFD-like detection, using antibodies, usually results in weak positive test lines at low antigen concentrations [44]. However, in the field of allergen detection, especially low levels of the respective allergenic food should be clearly detectable.…”

Scientific Opinion on the evaluation of allergenic foods and food ingredients for labelling purposes

“…17.6.3.3. Brazil nut, walnut, pistachio, pecan, macadamia and cashew nut A specific real-time PCR method for the detection of Brazil nut in processed food was compared with a commercially available qualitative lateral flow device (Röder et al, 2010), showing a LOD ≤ 5 mg/kg in spiked foods.…”

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

“…) as well as the diversity of processing treatments applied by the food industry, the preparation of calibration standards for each type of product is not feasible from a practical point of view. Thus, accurate quantification would require different standard curves for different products, according to their composition and processing conditions, as has been remarked on by previous studies (Fumière et al 2006;Koppelman & Hefle 2006;Piknova et al 2008;Röder et al 2010;Rojas et al 2010;Köppel et al 2012). The main limiting factor pointed out by these authors is the lack of knowledge of the food product composition, and processing conditions of the analysed samples, which prevents the construction of appropriate calibration standards.…”