Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection
Abstract
:1. Introduction
2. Results and Discussion
2.1. Primer Design and Primary Verification by PCR
2.2. Verification of Designed Primers for the Specificity of RPA–LFA
2.3. Sensitivity of RPA–LFA
2.4. Verification of the RPA–LFA with Meat Samples
2.5. Comparison of the Developed Technique to Control Chicken or Pig Adulteration and Other RPA-Based Assays
3. Materials and Methods
3.1. Reagents
3.2. Meat Samples
- Mix N1: 5% pig; 4.4% chicken; 90.6% bovine;
- Mix N2: 20% pig; 17.8% chicken; 62.2% bovine;
- Mix N3: 5% chicken muscle; 5% chicken skin; 90% pig;
- Mix N4: 20% chicken; 17.8% chicken skin; 62.2% pig.
- Additionally, mixes N3 and N4 were temperature-treated up to 72 °C. The sausages were of two types:
- Sausage N1, comprising 40% pig muscles, 25% pig fatback, and 35% bovine according to [65];
- Sausage N2, comprising 25% pig fatback and 75% bovine according to [66].
Meat | Target Sequence | Sensitivity of DNA | Adulteration, % | Detection Method | Time of Amplification/Time of LFA | LoD of PCR | Extraction Method | Extraction Time, Min | Ref. |
---|---|---|---|---|---|---|---|---|---|
Mangalica pig | Microsatellite locus | 0.17 ng/µL = 50 copies/reaction (1 copy/µL) | ND | LFA of RPA-nfo product | 30/5 | NA | Wizard® kit (Promega, Madison, WI, USA) ǁ DNAreleasy® (Nippon Genetics Europe, Düren, Germany) ǁ crude homogenization in water | >180 ǁ 15 ǁ <5 | [48] |
Chicken | D-loop | 104 copies/µL = 20 pg total DNA/reaction | 1 | SYBR Green I coloration | 30 | 100 copies/µL | Universal Genomic kit (CWBIO, Taizhou, China) | 85–205 | [45] |
Pig | 103 copies/µL = 20 pg total DNA/reaction | 1 | 100 copies/µL | ||||||
Pig | ND2 | 1.23 pg total DNA/reaction = 10 copies/reaction | 0.1 | Real-time fluorescence by mobile equipment | 15 | NA | QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) | 20 | [44] |
Pig | D-loop | 10 pg total pig DNA | 1 | Probe hybridization (with stage at 95 °C for 5 min) followed by LFA | 40/8 | DNeasy blood and tissue kit (Qiagen, Hilden, Germany) | 20–60 | [46] | |
Chicken | NDL4 | 10 copies (plasmid)/µL, 20 pg total DNA/µL | 1 | LFA of RPA-nfo product | 20/4 | Pos/neg test | gDNA extraction kit (Tiangen, Beijing, China) | 60 | [47] |
Pig | ND1 | 10 copies (plasmid)/µL, 20 pg total DNA/µL | |||||||
Chicken | Cyt B | 0.2 pg total DNA/µL = 20 copies/ µL | 5 | LFA of TwisDx basic products | 20/10 | 0.1 pg total DNA/µL | Salt method ǁ Crude homogenization | 120 ǁ <3 | This study |
Pig | 0.2 pg total DNA/µL = 2 copies/µL | 0.1 pg total DNA/µL |
3.3. Meat Processing and DNA Extraction
3.4. Primer Design
3.5. Synthesis of Cytochrome b Genes
3.6. Quantitative Real-Time PCR
3.7. Preparation of Lateral Flow Test Strips
3.8. RPA–LFA Test
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Species | Name | Sequences (5′ to 3′) | Length | Modification of 5′ |
---|---|---|---|---|
Galus galus | F1c | TCACATCGGACGAGGCCTA | 19 | Biotin |
R1c | GGAATGGGGTGAGTATGAGAGTT | 23 | FAM | |
F2c | TCACATCGGACGAGGCCTATACTAC | 25 | Biotin | |
F3c | CCTATTAGCAGTCTGCCTCATGACC | 25 | Biotin | |
R3c | GAGGCGCCGTTTGCGTGGAGATTCC | 25 | FAM | |
F4c | CTTCAAAGACATTCTGGGCTTAACTC | 26 | Biotin | |
R4c | ATTTTGTTTTCTAGTGTTCCGATTGT | 26 | FAM | |
Sus scrofa | F1p | GACCTCCCAGCTCCATCAAACATCTCATCATGATGAAA | 38 | Biotin |
R1p | GCTGATAGTAGATTTGTGATGACCGTA | 27 | FAM | |
F2p | AACAACAGCTTTCTCATCAGTTACA | 25 | Biotin | |
F3p | AAATTACGGATGAGTTATTCGCTATC | 26 | Biotin | |
R3p | GTGCAGGAATATGAGATGTACGGCT | 25 | FAM | |
F4p | AAAGACATTCTAGGAGCCTTATTTA | 25 | Biotin | |
R4p | TAGGATGGAGGCTACTAGGGCCAAC | 25 | FAM | |
F5p | AGCCTCCATCCTAATCCTAATTTTA | 25 | Biotin | |
R6p | ATAGGTTGTTTTCGATGATGCTAGTG | 26 | FAM |
Chicken (G. galus) | |||||
---|---|---|---|---|---|
F1c | F2c | F3c | F4c | ||
R1c | 431 | 431 | 614 | 44 | |
R3c | NA | NA | 159 | NA | |
R4c | 840 | 840 | 1023 | 453 | |
Pig (S. scrofa) | |||||
F1p | F2p | F3p | F4p | F5p | |
R1p | 348 | 279 | 273 | NA | NA |
R3p | 546 | 427 | 385 | NA | NA |
R4p | 840 | 721 | 679 | 219 | NA |
R6p | 107 | 953 | 911 | 415 | 246 |
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Ivanov, A.V.; Popravko, D.S.; Safenkova, I.V.; Zvereva, E.A.; Dzantiev, B.B.; Zherdev, A.V. Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection. Molecules 2021, 26, 6804. https://doi.org/10.3390/molecules26226804
Ivanov AV, Popravko DS, Safenkova IV, Zvereva EA, Dzantiev BB, Zherdev AV. Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection. Molecules. 2021; 26(22):6804. https://doi.org/10.3390/molecules26226804
Chicago/Turabian StyleIvanov, Aleksandr V., Demid S. Popravko, Irina V. Safenkova, Elena A. Zvereva, Boris B. Dzantiev, and Anatoly V. Zherdev. 2021. "Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection" Molecules 26, no. 22: 6804. https://doi.org/10.3390/molecules26226804
APA StyleIvanov, A. V., Popravko, D. S., Safenkova, I. V., Zvereva, E. A., Dzantiev, B. B., & Zherdev, A. V. (2021). Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection. Molecules, 26(22), 6804. https://doi.org/10.3390/molecules26226804