cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.)
Abstract
1. Introduction
2. Materials and Methods
2.1. Samples
2.2. Model Samples
2.3. DNA Isolation
2.4. In Silico Analysis of Actin and cor Genes
2.5. Primers and Probes
Marker | Oligonucleotide † | Sequence (5′-3′) | Size [bp] | Reference |
---|---|---|---|---|
COR1 | COR1_F | CCTTGTATAAATATCCCCGGA | 207 | This work |
COR1_R | TCTGATTATGCCCTTATTCAAC | |||
COR1_P | FAM-AGTTGTTTCCATTTTTGGAGT CAAGTTGAGACA-BHQ-1 | |||
Actin | Act-L_F | CAAGCAGCATGAAGATCAAGGT | ~103 | [29,30] |
Act-L_R | CACATCTGTTGGAAAGTGCTGAG | |||
Act-L_P | HEX-CCTCCAATCCAGACACTGTA CTTYCTCTC-BHQ-1 | |||
Actin-Z_F | CCCTGGAATTGCTGATAGGATGA | 150 ‡ | This work | |
Actin-Z_P | HEX-ATCACAGCTCTTGCACCAAGCAG CATGAAG-BHQ-1 | |||
Myostatin | MY_F | TTGTGCAAATCCTGAGACTCAT | 97 | [31] |
MY_R | ATACCAGTGCCTGGGTTCAT |
2.6. End-Point PCR
2.7. Quantitative Real-Time PCR (qPCR)
2.8. Droplet Digital PCR (ddPCR)
2.9. Multiplex PCR
2.10. Validation of Designed PCR Assays
2.10.1. Specificity
2.10.2. Sensitivity
3. Results
3.1. Actin Gene as a Reference Marker and Control of DNA Amplification
3.2. cor Gene as a Marker for P. somniferum Discrimination
3.3. Comparison of Three PCR Types
3.4. Multiplexing of the PCR Assays
3.5. Analysis of Heat-Treated Seeds and Real Food Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Family | Genus | Species | Common Name | Source |
---|---|---|---|---|
Papaveraceae | Argemone | A. mexicana | Mexican poppy | Seeds, leaves |
Papaver | P. bracteatum | Iranian poppy, Persian poppy | Leaves | |
P. commutatum | Caucasian scarlet poppy | Leaves | ||
P. glaucum | Tulip poppy | Seeds, leaves | ||
P. nudicaule | Iceland poppy | Seeds, leaves | ||
P. orientale | Oriental poppy | Seeds, leaves | ||
P. rhoeas | Common poppy | Seeds, leaves | ||
P. somniferum | Opium poppy | Seeds, leaves, stems, poppyheads | ||
Amaranthaceae | Amaranthus | (not specified) | Amaranth | Seeds |
Asteraceae | Helianthus | H. annuus | Sunflower | Seeds |
Brassicaceae | Brassica | B. napus | Rape | Leaves |
Sinapsis | S. alba | White mustard | Leaves | |
Fabaceae | Glycine | G. max | Soya-bean | Leaves |
Medicago | M. sativa | Alfalfa | Seeds, leaves | |
Linaceae | Linum | L. usitatissimum | Common flax | Seeds |
Poaceae | Avena | A. sativa | Common oat | Grain, leaves |
Hordeum | H. vulgare | Six-rowed barley | Grain, leaves | |
Oryza | O. sativa | Burgundy rice | Leaves | |
Secale | S. cereale | Cereal rye | Grain, leaves | |
Sorghum | S. bicolor | Great Millet, Sorghum | Seeds, leaves | |
Triticum | T. aestivum | Bread wheat | Grain, leaves | |
Zea | Z. mays | Maize | Grain, leaves | |
Solanaceae | Capsicum | C. annuum | Sweet Pepper | Leaves, pod |
Solanum | S. tuberosum | Potato | Tissue | |
S. lycopersicum | Tomato | Leaves | ||
Bovidae | Bos | B. taurus | Cattle | Tissue |
Equidae | Equus | E. caballus | Horse | Tissue |
Phasianidae | Gallus | G. gallus | Chicken | Tissue |
Suidae | Sus | S. scrofa | Pig | Tissue |
Treatment Conditions | Type of Treatment | Poppy Seeds | |
---|---|---|---|
80 °C, 30 min | Baking | Whole | |
Ground | |||
100 °C, 2 min | Cooking | in water | Ground |
in milk with the addition of sugar | |||
180 °C, 30 min | Baking | Seeds after cooking in water | |
Seeds after cooking in milk | |||
Ground (without previous cooking) | |||
Whole (without previous cooking) | |||
Dried at room temperature; without heat treatment | Ground |
Group | Sample | DNA Amplification Control † | Amplification of cor Gene ‡ | |||
---|---|---|---|---|---|---|
PCR (All Tested Platforms) | PCR | qPCR (EvaGreen/Probe) | ddPCR (Probe) | |||
Papaveraceae | Opium poppy | Aplaus | + | + | + | + |
Bergamon | + | + | + | + | ||
Buddha | + | + | + | + | ||
Major | + | + | + | + | ||
Maraton | + | + | + | + | ||
Opal | + | + | + | + | ||
Opex | + | + | + | + | ||
Peony | + | + | + | + | ||
Postomi | + | + | + | + | ||
Caucasian scarlet poppy | + | − | − | − | ||
Common poppy | + | − | − | − | ||
Iceland poppy | + | − | − | − | ||
Iranian or Persian Poppy | + | − | − | − | ||
Mexican Poppy | + | − | − | − | ||
Oriental Poppy | + | − | − | − | ||
Tulip poppy | + | − | − | − | ||
Other plant species | Alfalfa | + | − | − | − | |
Amaranth | + | − | − | − | ||
Barley | + | − | − | −/+ | ||
Flax | + | − | − | − | ||
Maize | + | − | − | − | ||
Mustard | + | − | +/− | −/+ | ||
Oat | + | − | − | − | ||
Oilseed rape | + | − | − | − | ||
Pepper | + | − | +/− | − | ||
Potato | + | − | +/− | − | ||
Rice | + | − | − | − | ||
Rye | + | − | − | − | ||
Sorghum | + | − | − | − | ||
Soya | + | − | − | |||
Sunflower | + | − | − | − | ||
Tomato | + | − | − | − | ||
Wheat | + | − | +/− | − | ||
Animal species | Cattle | + | − | − | − | |
Horse | + | − | N | − | ||
Chicken | + | − | − | − | ||
Pig | + | − | − | − |
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Čermáková, E.; Svoboda, P.; Ovesná, J.; Vašek, J.; Demnerová, K.; Zdeňková, K. cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.). Foods 2024, 13, 1432. https://doi.org/10.3390/foods13101432
Čermáková E, Svoboda P, Ovesná J, Vašek J, Demnerová K, Zdeňková K. cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.). Foods. 2024; 13(10):1432. https://doi.org/10.3390/foods13101432
Chicago/Turabian StyleČermáková, Eliška, Pavel Svoboda, Jaroslava Ovesná, Jakub Vašek, Kateřina Demnerová, and Kamila Zdeňková. 2024. "cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.)" Foods 13, no. 10: 1432. https://doi.org/10.3390/foods13101432
APA StyleČermáková, E., Svoboda, P., Ovesná, J., Vašek, J., Demnerová, K., & Zdeňková, K. (2024). cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.). Foods, 13(10), 1432. https://doi.org/10.3390/foods13101432