LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food

Soybean (Glycine max) allergy can be life threatening. A lack of causative immunotherapy of soybean allergy makes soybean avoidance indispensable. Detection methods are essential to verify allergen labeling and unintentional allergen cross contact during food manufacture. Here, we aimed at evaluating our previously described primers for loop-mediated isothermal amplification (LAMP) of multicopy gene ORF160b, combined with a lateral flow dipstick (LFD)-like detection, for their performance of soybean detection in complex food matrices. The results were compared with those obtained using quantitative real-time Polymerase Chain Reaction (qPCR) as the current standard of DNA-based allergen detection, and antibody-based commercial lateral flow device (LFD) as the current reference of protein-based rapid allergen detection. LAMP-LFD allowed unequivocal and reproducible detection of 10 mg/kg soybean incurred in three representative matrices (boiled sausage, chocolate, instant tomato soup), while clear visibility of positive test lines of two commercial LFD tests was between 10 and 102 mg/kg and depending on the matrix. Sensitivity of soybean detection in incurred food matrices, commercial retail samples, as well as various processed soybean products was comparable between LAMP-LFD and qPCR. The DNA-based LAMP-LFD proved to be a simple and low-technology soybean detection tool, showing sensitivity and specificity that is comparable or superior to the investigated commercial protein-based LFD.

PSI-10 Genetic parameters for soybean allergy response in divergently-selected pigs

Genetically selecting for improved animal resilience has become of great interest in modern livestock breeding. In this context, piglets experience a transitory allergic response to soybean proteins that has a negative impact on their growth and welfare. The main objective of this study was to determine whether soybean allergy response is a heritable trait, using divergently selected pig lines. The base generation pigs were generated from nine sire lines (primarily Yorkshire and Landrace) and two dam sources (Yorkshire × Chester White F1 and Landrace × Yorkshire F1). Soybean allergic response was measured, following a skin test protocol, on pigs that received a diet containing 28% soybean meal for 21 consecutive days post-weaning. The skin test was performed with an intradermal injection of soybean protein. Wheel and flare scores (ranging from 0 to 3) were assigned approximately 60 minutes post-injection. Based on the skin test total scores (i.e., the sum of the wheal and flair scores, ranging from 0 to 6), animals were selected to create two (a high and a low) soybean protein reacting lines, and bred within lines for nine generations. Phenotypes used in this study were measured on a total of 5,505 animals born from generations one to nine, in both high and low reacting lines. Significant systematic effects (P < 0.05) included in the statistical model were birth year, birth season, sex, and weaning weight (as a covariate). Genetic parameters for the skin test were estimated using a pedigree-based relationship matrix containing 9,201 animals, and a threshold animal mixed model. The genetic variance estimated for soybean allergy was 0.091, indicating that there is genetic variability in this trait. The heritability estimated for soybean allergy response was 0.139, which indicates that food allergy response is a heritable trait and can be improved through selective breeding.

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)

The soybean (Glycine max) has been recognized as a frequent elicitor of food allergy worldwide. A lack of causative immunotherapy of soybean allergy makes soybean avoidance essential. Therefore, sensitive and specific methods for soybean detection are needed to allow for soybean verification in foods. Loop-mediated isothermal amplification (LAMP) represents a rapid and simple DNA-based detection method principally suitable for field-like applications or on-site analytical screening for allergens during the manufacturing of foods. This work describes the systematic development and selection of suitable LAMP primers based on soybean multicopy genes. The chemistry applied allows for a versatile detection of amplified DNA, using either gel electrophoresis, fluorescence recording, or a simple Lateral Flow Dipstick (LFD). LAMP based on the ORF160b gene was highly specific for the soybean and may allow for a detection level equivalent to approximately 10 mg soy per kg food. Various soybean cultivars were detectable at a comparable level of sensitivity. LAMP combined with LFD-like detection facilitates a simple, highly specific and sensitive detection of the soybean without the need for expensive analytical equipment. In contrast to the majority of antibody-based methods for soybean detection, all identified primer sequences and optimized protocols are disclosed and broadly available to the community.