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
APA StyleAllgöwer, S. M., Hartmann, C. A., Lipinski, C., Mahler, V., Randow, S., Völker, E., & Holzhauser, T. (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(12), 1741. https://doi.org/10.3390/foods9121741