LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Retail Foods
2.2. Soybean Incurred Food Matrices
2.2.1. Sausages
2.2.2. Chocolate
2.2.3. Instant Tomato Soup
2.3. Commercial Protein-Based LFD
2.4. DNA Extraction and Purification
2.5. Oligonucleotides
2.6. Eukaryotic qPCR
2.7. Generation of Plasmid Standards for Lectin qPCR
2.8. Lectin qPCR
2.9. ORF160b LAMP-LFD
2.10. Replicate Analysis of Food Samples
2.11. Statistical Analysis
3. Results
3.1. Specificity of the Methods
3.2. Detectability of Soy Products
3.3. Determination of Method Sensitvity in Soybean-Incurred Food Matrices
3.3.1. Verification of Various Levels of Incurred Soybean
3.3.2. Comparison of Sensitivity of ORF160b LAMP-LFD and Commercial Protein-Based LFDs
3.4. Analyses of Retail Food Samples
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Investigation | ORF160b LAMP-LFD | Lectin qPCR | Commercial LFD 1 |
---|---|---|---|
Specificity | Allgöwer et al. 2020 [18] | n.d. 2 | 1 rxn 3, 1:10 dilution (1 extract × 1 rxn) |
Processed soy products | 4 rxns, 1:104 dilution (2 extracts × 2 rxns) | 4 rxns, 1:104 dilution (2 extracts × 2 rxns) | 1 rxn, 1:104 dilution (1 extract × 1 rxn) |
Sensitivity in incurred food matrices | 8 rxns, undiluted (2 extracts × 4 rxns) | 8 rxns, undiluted (2 extracts × 4 rxns) | 1 rxn, undiluted (1 extract × 1 rxn) |
Retail foods | 4 rxns, undiluted (2 extracts × 2 rxns) | 4 rxns, undiluted (2 extracts × 2 rxns) | 1 rxn, undiluted (1 extract × 1 rxn) |
Sample No. | Soy Product | Protein Content (%) | ORF160b LAMP-LFD | lectin qPCR | LFD1 | LFD2 |
---|---|---|---|---|---|---|
P-1 | TVP 1 | 56.7 | + | + | + | − |
P-2 | TVP 1 | 67.4 | (+) | (+) | + | − |
P-3 | soy flakes | ~50 2 | + | + | + | − |
P-4 | soy flakes | ~50 2 | + | + | + | + |
P-5 | soy protein concentrate | 71.8 | + | + | + | + |
P-6 | defatted soy flour | ~50 2 | + | + | + | + |
P-7 | soy semolina | ~50 2 | + | + | + | + |
P-8 | tofu | 13.5 | + | (+) | + | − |
18 | Yellow soybeans Schoenenberger® Hensel® | 38 | + | + | + | + |
Method | Food Matrix | Level of Incurred Soybean per Matrix (mg/kg) | ||||||
---|---|---|---|---|---|---|---|---|
105 | 104 | 103 | 102 | 101 | 100 | 0 1 | ||
lectin qPCR | sausage | + | + | + | + | + | (+) | − |
chocolate | + | + | + | + | + | + | − | |
tomato soup | + | + | + | + | + | (+) | − | |
ORF160b LAMP-LFD | sausage | + | + | + | + | + | (+) | − |
chocolate | + | + | + | + | + | + | − | |
tomato soup | + | + | + | + | + | (+) | − | |
LFD1 | sausage | n.d. 2 | n.d. | + | + | (+) | − | − |
chocolate | n.d. | n.d. | + | + | + | (+) | − | |
tomato soup | n.d. | n.d. | + | + | + | (+) | (+) | |
LFD2 | sausage | n.d. | n.d. | + | + | + | − | − |
chocolate | n.d. | n.d. | + | + | − | − | − | |
tomato soup | n.d. | n.d. | + | + | + | − | − |
Sample No. | Product Description | Soy: Ingredient Labeling (IL)/Precautionary Allergen Labeling (PAL) | Interpretation of IL/PAL | Lectin qPCR | ORF160b LAMP-LFD | LFD1 | LFD2 |
---|---|---|---|---|---|---|---|
R-1 | fine biscuit assortment | IL: soy flour, emulsifier: soy lecithin/PAL: none | contains soy | + | + | + | + |
R-2 | Asia style rice crackers | IL: soy sauce (soybeans, …)/PAL: contains soy | contains soy | − | − | − | − |
R-3 | sponge cake vanilla and raspberry taste with cocoa compound coating | IL: soybean, soy flour, emulsifier: soy lecithin/PAL: contains soy | contains soy | + | + | + | + |
R-4 | vegetable balls | IL: 10% plant protein (soy protein, wheat protein), hydrolyzed soy protein/PAL: none | contains soy | + | + | + | + |
R-5 | meat free, lactose free sausage-type bar for barbecue | IL: 12% soy protein/PAL: none | contains soy | + | + | ± 1 | + |
R-6 | cereal bar with chocolate | IL: none/PAL: none | no indication of soy presence | + | + | (+) | − |
R-7 | milk chocolate with puffed rice | IL: emulsifier: soy lecithin/PAL: none | contains soy | (+) | + | (+) | − |
R-8 | shortbread with milk chocolate and chopped almonds | IL: soy flour, emulsifier: soy lecithin/PAL: none | contains soy | + | + | + | + |
R-9 | hazelnut cookies | IL: none/PAL: produced in a facility, where soy is also processed | may contain soy | (+) | (+) | − | − |
R-10 | oat porridge | IL: none/PAL: may contain traces of soy | may contain soy | − | − | − | − |
R-11 | cashews roasted and salted | IL: none/PAL: none | no indication of soy presence | − | − | − | − |
R-12 | Cereal-potato-snack Western style flavor | IL: none/PAL: none | no indication of soy presence | (+) | − | (+) | − |
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Allgöwer, S.M.; Hartmann, C.A.; Lipinski, C.; Mahler, V.; Randow, S.; Völker, E.; Holzhauser, T. LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food. Foods 2020, 9, 1741. https://doi.org/10.3390/foods9121741
Allgöwer SM, Hartmann CA, Lipinski C, Mahler V, Randow S, Völker E, Holzhauser T. LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food. Foods. 2020; 9(12):1741. https://doi.org/10.3390/foods9121741
Chicago/Turabian StyleAllgöwer, Stefanie M., Chris A. Hartmann, Clarissa Lipinski, Vera Mahler, Stefanie Randow, Elke Völker, and Thomas Holzhauser. 2020. "LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food" Foods 9, no. 12: 1741. https://doi.org/10.3390/foods9121741