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://0-doi-org.brum.beds.ac.uk/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://0-doi-org.brum.beds.ac.uk/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://0-doi-org.brum.beds.ac.uk/10.3390/foods9081049