Sensitive detection of allergens targeting multi-copy genes

An International Standard based Real-Time PCR detection portfolio.

 

DNA based technology is a relatively new approach for the detection of food allergens, a field traditionally dominated by immunoassays.  

DNA methods are indirect methods to detect food allergens as they do not detect the allergenic protein itself, but the DNA sequences from the allergenic food ingredient.  

These DNA based analytical methods are fast, sensitive, and provide specific detection of the targeted DNA. Since the publication of EN15634 and EN15842 (EN 15634-1:2019; EN 15842:2019) back in 2009, the use of Real-Time PCR in allergen detection become attractive given the evident advantages versus ELISA.  

Nowadays in Germany and Japan, Real-time PCR methods have been established as official analytical methods for allergen detection (Holzhauser et al. 2020; BVL L44.00-11 2013; Waiblinger and Schulze 2018; Shoji et al. 2018).  

The main advantage of DNA-based methods over protein-based methodologies is that the target DNA is efficiently extracted from raw and cooked products and is only slightly affected by the heating process. DNA typically remains only fragmentated, but still detectable after being exposed to the cooking temperatures of most foods. As stated in EN15842 “a positive result of a DNA based method will correlate with the presence of allergenic proteins”. In contrast, attention must be given to negative results using targeted protein-based methods (e.g. ELISA) because food processing might impair antibody detection of allergens, whilst the capacity for the proteins to elicit an allergic response still exists. Some limitations exist in the use of PCR for the detection of allergens in food, especially for milk and egg, and when food contains added purified protein extracts.  

... specificity and sensitivity along with an efficient DNA extraction process is paramount for the success of a molecular allergen detection method.

In most country’s food safety regulations, it states that allergens must be “absent”. For food safety scientists this translates to allergens being “undetectable” by analytical means. Hence, although the possibility of quantitation is valuable, specificity and sensitivity along with an efficient DNA extraction process is paramount for the success of a molecular allergen detection method.  Therefore, Generon SPECIALfinder MC allergen detection kits were designed with this successful requirement in mind.  

The careful selection of the specific target was an essential part of our R&D effort. Although allergen-coding sequences are attractive targets and recommended for quantification purposes, they are present in low copy number in the species genome thus compromising the sensitivity of the method. In compliance with EN15634 we preferred to target multi-copy genes (i.e. mitochondrial, chloroplast or repetitive sequences as ITS) in order to detect an allergenic ingredient. With this approach it is possible to achieve a sensitivity comparable to ELISA (noteworthy proteins are present in multiple copies inside cells).  

Figure 1 shows the difference in sensitivity between SPECIALfinder MC Mustard and EN15634-5 proposed mustard Real-Time PCR detection design.

Figure 1: amplification curve of two DNA extracts (total DNA loaded 200 ng/ reactio