Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products
Abstract
1. Introduction
2. Material and Methods
2.1. Material
2.1.1. Chemical Material
2.1.2. Sample Material
2.2. Methods
2.2.1. Bioinformatics
2.2.2. DNA Isolation
2.2.3. Real-Time PCR
Reaction Set-Up
Templates
2.2.4. Calculation
Method A: Quantification with Reference Material
Method B: Quantification with Matrix-Specific Multiplication Factors
Method C: Quantification with Internal Reference Sequence
2.2.5. Statistical Analysis
3. Results
3.1. Bioinformatics
3.2. Development of One Triplex and One Duplex Real-Time PCR System
3.3. Quantification
3.3.1. Method A: Quantification with Reference Material
3.3.2. Method B: Quantification with Matrix-Specific Multiplication Factors
3.3.3. Method C: Quantification with an Internal Reference Sequence
3.3.4. Comparison
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Amount of Meat Added (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
Poultry Species | S1 | S2 | S3 | S4 | S5 | U1 | U2 | U3 | U4 | U5 |
Chicken | 1.0 | 0.0 | 69.0 | 25.0 | 5.0 | 2.0 | 0.5 | 57.5 | 32.0 | 8.0 |
Guinea fowl | 25.0 | 5.0 | 1.0 | 0.0 | 69.0 | 32.0 | 8.0 | 2.0 | 0.5 | 57.5 |
Pheasant | 0.0 | 69.0 | 25.0 | 5.0 | 1.0 | 0.5 | 57.5 | 32.0 | 8.0 | 2.0 |
Quail | 5.0 | 1.0 | 0.0 | 69.0 | 25.0 | 8.0 | 2.0 | 0.5 | 57.5 | 32.0 |
Turkey | 69.0 | 25.0 | 5.0 | 1.0 | 0.0 | 57.5 | 32.0 | 8.0 | 2.0 | 0.5 |
Multiplex Real-Time PCR | Animal Species | Gene | Code | DNA Sequence 5′–3′ | Concentration (µM) | Reference |
---|---|---|---|---|---|---|
C-G-P | Chicken | Cyt b | C-for | AGC AAT TCC CTA CAT TGG ACA CA | 0.20 | [27] |
C-rev | GAT GAT AGT AAT ACC TGC GAT TGC A | 0.20 | ||||
C-probe | JOE-CAG TCG ACA ACC CAA CCC TTA CCC GAT TC-BHQ1 | 0.08 | [32] | |||
Guinea fowl | Cyt b | G-for | GCA TAC GCC ATC CTC CGC TC | 0.20 | [33] | |
G-rev | GCT GCC CAC TCA GGT TAG A | 0.20 | ||||
G-probe | DY682-TGG AGG CGT ACT AGC ACT AGC AGC CTC CG-BHQ2 | 0.08 | [32] | |||
Pheasant | Cyt b | P-for | GAG ACA TGA AAC ACT GGA G | 0.20 | [33] | |
P-rev | CAG GTC CAT TCT ACC AAG G | 0.20 | ||||
P-probe | ATTO633-CGT CCT ACT CCT CAC ACT CAT AGC AAC C-BHQ2 | 0.08 | [32] | |||
Q-T | Quail | Cyt b | Q-for | TGT ACC CTA CAT CGG CCA AAC C | 0.20 | [33] |
Q-rev | GTC AGA TGA GAT TCC TAA TGG G | 0.20 | ||||
Q-probe | FAM-CCT ACC CTA ACC CGA TTC TTC GCC CTC C-BHQ1 | 0.10 | [32] | |||
Turkey | Cyt b | T-for | CAC TCT TGC ATT CTC TTC TGT GG | 0.20 | [33] | |
T-rev | GGA GGT TAT GGA GGA GTC AAC | 0.20 | ||||
T-probe | ROX-CCT ACA CAT GCC GAA ACG TAC AAT ACG-BHQ2 | 0.08 | [32] | |||
ALL | Eukarya | 12S rRNA | 12S-for | AAA CTG GGA TTA GAT ACC CCA CTA TG | 0.3 | This work |
12S-rev | AGA ACA GGC TCC TCT AGG TGG | 0.3 | ||||
12S-probe | FAM-AGA ACT ACG AGC ACA AAC GCT TAA AAC TCT A-BHQ1 | 0.2 |
DNA | Triplex C-G-P | Duplex Q-T | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Chicken | Guinea Fowl | Pheasant | Quail | Turkey | ||||||
Asparagus | 32.28 | 32.96 | - | - | - | - | - | - | - | - |
Beef | 32.20 | - | - | - | - | - | - | - | - | - |
Chicken | 15.21 | 15.47 | - | - | 31.27 | 31.06 | - | - | - | - |
Deer | - | - | - | - | - | - | 34.74 | 34.26 | - | - |
Duck | - | - | - | - | 34.64 | - | - | - | - | - |
Goose | - | - | - | - | 29.54 | 29.22 | - | - | 34.23 | - |
Guinea fowl | 32.95 | 34.77 | 16.40 | 16.71 | - | - | - | - | - | - |
Kangaroo | - | - | - | - | - | - | 32.33 | 31.46 | - | - |
Mace | 32.13 | - | - | - | 29.53 | - | - | - | - | - |
Ostrich | - | - | 31.01 | 30.59 | 30.49 | 30.12 | 30.94 | 31.20 | - | - |
Pheasant | - | - | - | - | 14.70 | 14.73 | - | - | - | - |
Quail | - | - | - | - | - | - | 17.69 | 17.73 | - | - |
Turkey | - | - | - | - | - | - | - | - | 15.86 | 16.00 |
Wild boar | - | - | - | - | - | - | - | - | 34.80 | - |
Blank value | 32.66 | - | 32.36 | 32.33 | 29.13 | 29.73 | 32.23 | 31.35 | 31.71 | 32.38 |
Actual (% w/w) | Low Temperature | High Temperature | ||||||
---|---|---|---|---|---|---|---|---|
Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | |
Chicken | ||||||||
0.5 | 0.28 | 0.04 | 14.41 | −43.33 | 0.33 | 0.08 | 24.49 | −33.33 |
2.0 | 1.88 | 0.26 | 14.01 | −5.83 | 2.43 | 0.27 | 11.23 | 21.67 |
8.0 | 6.82 | 0.54 | 7.95 | −14.79 | 7.22 | 1.64 | 22.71 | −9.79 |
32.0 | 25.22 | 3.14 | 12.47 | −21.20 | 36.45 | 7.15 | 19.61 | 13.91 |
57.5 | 50.53 | 10.56 | 20.90 | −12.12 | 70.88 | 6.79 | 9.57 | 23.28 |
Guinea fowl | ||||||||
0.5 | 0.30 | 0.06 | 21.08 | −40.00 | 0.37 | 0.08 | 22.27 | −26.67 |
2.0 | 1.52 | 0.24 | 15.83 | −24.17 | 2.48 | 0.69 | 27.95 | 24.17 |
8.0 | 8.45 | 0.73 | 8.62 | 5.63 | 10.30 | 2.42 | 23.48 | 28.75 |
32.0 | 26.63 | 3.93 | 14.74 | −16.77 | 43.10 | 8.06 | 18.69 | 34.69 |
57.5 | 45.90 | 3.68 | 8.01 | −20.17 | 57.42 | 17.53 | 30.53 | −0.14 |
Pheasant | ||||||||
0.5 | 0.55 | 0.05 | 9.96 | 10.00 | 0.87 | 0.30 | 34.74 | 73.33 |
2.0 | 1.68 | 0.22 | 13.24 | −15.83 | 1.78 | 0.15 | 8.25 | −10.83 |
8.0 | 6.92 | 0.96 | 13.94 | −13.54 | 10.45 | 1.35 | 12.96 | 30.63 |
32.0 | 24.85 | 3.93 | 15.81 | −22.34 | 36.02 | 6.30 | 17.49 | 12.55 |
57.5 | 55.53 | 3.66 | 6.59 | −3.42 | 57.60 | 13.94 | 24.19 | 0.17 |
Quail | ||||||||
0.5 | 0.47 | 0.16 | 34.99 | −6.67 | 0.70 | 0.15 | 22.13 | 40.00 |
2.0 | 2.03 | 0.20 | 9.67 | 1.67 | 2.22 | 0.43 | 19.44 | 10.83 |
8.0 | 7.90 | 0.88 | 11.12 | −1.25 | 9.68 | 2.41 | 24.91 | 21.04 |
32.0 | 28.83 | 7.56 | 26.23 | −9.90 | 29.17 | 2.35 | 8.05 | −8.85 |
57.5 | 50.32 | 10.41 | 20.69 | −12.49 | 95.07 | 37.50 | 39.45 | 65.33 |
Turkey | ||||||||
0.5 | 0.52 | 0.16 | 31.01 | 3.33 | 0.45 | 0.05 | 12.17 | −10.00 |
2.0 | 1.68 | 0.19 | 11.53 | −15.83 | 2.03 | 0.30 | 14.81 | 1.67 |
8.0 | 4.95 | 0.23 | 4.56 | −38.13 | 7.42 | 0.80 | 10.83 | −7.29 |
32.0 | 29.05 | 2.81 | 9.68 | −9.22 | 32.60 | 5.69 | 17.45 | 1.88 |
57.5 | 49.42 | 12.04 | 24.37 | −14.06 | 54.68 | 5.37 | 9.82 | −4.90 |
Temperature | Batch | Chicken | Guinea Fowl | Pheasant | Quail | Turkey |
---|---|---|---|---|---|---|
Low | A | 1.16 | 1.19 | 0.68 | 3.02 | 1.19 |
B | 1.44 | 1.49 | 1.12 | 4.61 | 1.17 | |
Mean | 1.30 | 1.34 | 0.90 | 3.82 | 1.18 | |
High | A | 0.24 | 0.12 | 0.09 | 0.31 | 0.61 |
B | 0.23 | 0.09 | 0.10 | 0.29 | 0.42 | |
Mean | 0.24 | 0.11 | 0.09 | 0.30 | 0.52 |
Actual (% w/w) | Low Temperature | High Temperature | ||||||
---|---|---|---|---|---|---|---|---|
Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | |
Chicken | ||||||||
0.5 | 0.40 | 0.05 | 11.70 | −19.33 | 0.35 | 0.07 | 19.07 | −29.67 |
2.0 | 2.85 | 0.20 | 6.94 | 42.58 | 2.71 | 0.41 | 15.24 | 35.42 |
8.0 | 10.23 | 0.55 | 5.39 | 27.83 | 10.33 | 0.74 | 7.13 | 29.10 |
32.0 | 34.77 | 1.90 | 5.47 | 8.65 | 31.76 | 2.37 | 7.47 | −0.74 |
57.5 | 60.68 | 2.49 | 4.10 | 5.52 | 62.27 | 4.51 | 7.25 | 8.30 |
Guinea fowl | ||||||||
0.5 | 0.44 | 0.06 | 14.08 | −13.00 | 0.37 | 0.08 | 21.66 | −26.67 |
2.0 | 1.80 | 0.23 | 12.61 | −10.25 | 1.81 | 0.09 | 4.71 | −9.58 |
8.0 | 7.75 | 0.73 | 9.45 | −3.10 | 7.73 | 1.11 | 14.42 | −3.35 |
32.0 | 29.86 | 2.96 | 9.91 | −6.68 | 32.29 | 2.84 | 8.81 | 0.90 |
57.5 | 51.62 | 4.15 | 8.04 | −10.22 | 51.76 | 2.72 | 5.26 | −9.98 |
Pheasant | ||||||||
0.5 | 0.74 | 0.19 | 25.55 | 47.33 | 0.69 | 0.17 | 24.82 | 38.00 |
2.0 | 2.50 | 0.33 | 13.21 | 24.75 | 2.00 | 0.28 | 14.08 | 0.00 |
8.0 | 9.25 | 1.60 | 17.33 | 15.65 | 8.50 | 1.64 | 19.33 | 6.19 |
32.0 | 31.41 | 3.44 | 10.96 | −1.83 | 31.11 | 4.83 | 15.53 | −2.79 |
57.5 | 63.42 | 5.17 | 8.16 | 10.30 | 63.70 | 5.12 | 8.03 | 10.79 |
Quail | ||||||||
0.5 | 0.43 | 0.10 | 22.58 | −13.67 | 0.45 | 0.06 | 13.19 | −10.33 |
2.0 | 1.81 | 0.22 | 11.95 | −9.75 | 1.90 | 0.19 | 9.94 | −5.17 |
8.0 | 8.90 | 0.70 | 7.83 | 11.21 | 9.54 | 1.06 | 11.10 | 19.23 |
32.0 | 35.00 | 4.17 | 11.93 | 9.36 | 35.42 | 2.81 | 7.95 | 10.68 |
57.5 | 53.81 | 3.19 | 5.93 | −6.42 | 57.99 | 4.04 | 6.96 | 0.86 |
Turkey | ||||||||
0.5 | 0.66 | 0.13 | 19.06 | 32.33 | 0.50 | 0.06 | 12.78 | −0.67 |
2.0 | 1.74 | 0.17 | 9.89 | −13.00 | 1.38 | 0.16 | 11.85 | −31.00 |
8.0 | 5.68 | 0.94 | 16.46 | −28.98 | 4.37 | 0.42 | 9.65 | −45.42 |
32.0 | 26.62 | 4.58 | 17.19 | −16.81 | 26.32 | 4.03 | 15.32 | −17.76 |
57.5 | 57.65 | 2.79 | 4.84 | 0.26 | 54.78 | 2.57 | 4.69 | −4.74 |
Actual (% w/w) | Low Temperature | High Temperature | ||||||
---|---|---|---|---|---|---|---|---|
Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | Mean Predicted (% w/w) a | SD b | CV (%) c | Bias (%) d | |
Chicken | ||||||||
0.5 | 0.69 | 0.11 | 16.37 | 37.67 | 0.57 | 0.21 | 37.23 | 14.00 |
2.0 | 2.06 | 0.20 | 9.65 | 3.08 | 2.03 | 0.32 | 15.90 | 1.58 |
8.0 | 7.38 | 2.64 | 35.76 | −7.79 | 7.08 | 0.68 | 9.56 | −11.56 |
32.0 | 26.22 | 2.48 | 9.47 | −18.05 | 26.61 | 4.78 | 17.97 | −16.84 |
57.5 | 72.45 | 9.83 | 13.56 | 25.99 | 73.04 | 9.12 | 12.49 | 27.02 |
Guinea fowl | ||||||||
0.5 | 0.21 | 0.04 | 21.00 | −57.67 | 0.25 | 0.07 | 30.08 | −50.67 |
2.0 | 1.87 | 0.42 | 22.56 | −6.50 | 2.09 | 0.50 | 23.74 | 4.75 |
8.0 | 10.85 | 3.17 | 29.24 | 35.56 | 9.36 | 1.99 | 21.25 | 17.02 |
32.0 | 33.86 | 7.46 | 22.03 | 5.82 | 32.24 | 5.27 | 16.36 | 0.73 |
57.5 | 57.65 | 17.42 | 30.21 | 0.26 | 60.87 | 13.56 | 22.28 | 5.85 |
Pheasant | ||||||||
0.5 | 0.67 | 0.15 | 21.82 | 34.67 | 0.67 | 0.05 | 7.32 | 34.67 |
2.0 | 1.85 | 0.76 | 41.35 | −7.50 | 1.75 | 0.25 | 14.53 | −12.58 |
8.0 | 6.99 | 0.66 | 9.46 | −12.62 | 7.22 | 0.85 | 11.76 | −9.79 |
32.0 | 31.17 | 3.02 | 9.68 | −2.59 | 34.09 | 2.49 | 7.31 | 6.52 |
57.5 | 53.74 | 6.71 | 12.49 | −6.54 | 52.72 | 8.00 | 15.18 | −8.31 |
Quail | ||||||||
0.5 | 0.35 | 0.16 | 47.44 | −31.00 | 0.41 | 0.11 | 26.76 | −18.00 |
2.0 | 1.92 | 0.57 | 29.44 | −3.92 | 1.65 | 0.30 | 18.42 | −17.50 |
8.0 | 7.33 | 1.64 | 22.44 | −8.40 | 8.21 | 1.02 | 12.38 | 2.67 |
32.0 | 31.10 | 5.97 | 19.20 | −2.83 | 32.02 | 6.33 | 19.76 | 0.06 |
57.5 | 57.61 | 8.82 | 15.31 | 0.19 | 66.62 | 10.82 | 16.24 | 15.87 |
Turkey | ||||||||
0.5 | 0.52 | 0.14 | 26.07 | 4.33 | 0.50 | 0.09 | 17.35 | 0.33 |
2.0 | 1.62 | 0.51 | 31.38 | −18.92 | 1.45 | 0.39 | 27.19 | −27.67 |
8.0 | 7.33 | 1.34 | 18.28 | −8.42 | 6.59 | 0.51 | 7.73 | −17.63 |
32.0 | 34.91 | 8.85 | 25.36 | 9.10 | 26.89 | 7.77 | 28.90 | −15.97 |
57.5 | 42.73 | 9.91 | 23.19 | −25.68 | 47.90 | 14.35 | 29.96 | −16.70 |
Method | Technical Summary | Bias | CV | Recovery Rate | Species |
---|---|---|---|---|---|
(% within Accepted Range) d | |||||
A a | Quantification with reference material | ||||
- Fast - Low costs | 80 | 100 | 68 | Chicken | |
60 | 80 | 65 | Guinea fowl | ||
80 | 90 | 80 | Pheasant | ||
80 | 70 | 77 | Quail | ||
90 | 90 | 85 | Turkey | ||
B b | Quantification with matrix-specific multiplication factors | ||||
- More time and more costs for establishment of multiplication-factors - Suited for repeated use | 50 | 100 | 70 | Chicken | |
90 | 100 | 93 | Guinea fowl | ||
80 | 90 | 82 | Pheasant | ||
100 | 100 | 97 | Quail | ||
60 | 100 | 73 | Turkey | ||
C c | Quantification with an internal reference sequence | ||||
- More time and more costs due to second real-time PCR assay - With inhibition control | 70 | 80 | 77 | Chicken | |
70 | 70 | 62 | Guinea fowl | ||
80 | 90 | 80 | Pheasant | ||
90 | 70 | 80 | Quail | ||
80 | 40 | 77 | Turkey |
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Dolch, K.; Andrée, S.; Schwägele, F. Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products. Foods 2020, 9, 1049. https://doi.org/10.3390/foods9081049
Dolch K, Andrée S, Schwägele F. Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products. Foods. 2020; 9(8):1049. https://doi.org/10.3390/foods9081049
Chicago/Turabian StyleDolch, Kerstin, Sabine Andrée, and Fredi Schwägele. 2020. "Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products" Foods 9, no. 8: 1049. https://doi.org/10.3390/foods9081049
APA StyleDolch, K., Andrée, S., & Schwägele, F. (2020). Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products. Foods, 9(8), 1049. https://doi.org/10.3390/foods9081049