Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples Collection and DNA Extraction
2.2. Design of Species-Specific Primers
2.3. Simplex and Multiplex PCR Assays
2.4. Sequencing of PCR Products
2.5. Tests of Specificity, Sensitivity and Reproducibility of Primers
3. Results
3.1. Specificity Assays of Designed Primers
3.2. Sensitivity of Multiplex PCR Assay
3.3. Reproducibility of Multiplex PCR Assay in Heat Processing Meat
3.4. Application of Multiplex PCR Assay on Commercial Meat Products
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primers | Genes | Sequence (5′-3′ Direction) | Amplicons (bp) | Reference or Source |
---|---|---|---|---|
Camel | NADH dehydrogenase subunit 4 | TCGCCAGCCAATCTCACCTCT | 726 | this study |
CATGGGCAACTATAAGGGTCGTA | ||||
Cattle | Cytochrome c oxidase subunit I | ATGAGCCCACCATATATTCACT | 610 | this study |
TGTCGTGGTTAAGTCTACAGTCA | ||||
Dog | NADH dehydrogenase subunit 5 | TGGCCTATTTAAAGTCCTTCCCT | 428 | this study |
CTAGTGCCAGGATGAAACCCAA | ||||
Pig | NADH dehydrogenase subunit 4 | AGCCTATCCATTCCTCATGCTTT | 332 | this study |
CTAGGTTTGTGAGGCTTGCTAC | ||||
Chicken | 16S rRNA | GAGTGCGTCAAAGCTCCCTC | 268 | this study |
GTTTGCCGAGTTCCTTCTGT | ||||
Sheep | NADH dehydrogenase subunit 2 | ATCCAATAGCCTCCATACTCA | 205 | this study |
ATTGATAGGGTTAGGATCAGGTC | ||||
Duck | Cytochrome c oxidase subunit III | TCCACGCCCTAACATTGACGATT | 163 | this study |
AAGGTGGATCCGATGATCACT | ||||
Eukaryotes | 12S rRNA | CAACTGGGATTAGATACCCCACTAT | 456 | [20] |
GAGGGTGACGGGCGGTGTGT | ||||
Eukaryotes | 16S rRNA | AAGACGAGAAGACCCTATGGA | 240 | [21] |
GATTGCGCTGTTATCCCTAGGGTA | ||||
Eukaryotes | 18S rRNA | AGGATCCATTGGAGGGCAAGT | 99 | [22] |
TCCAACTACGAGCTTTTTAACTGCA |
Products | Number | Labelled | Detected Species | Adulteration | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Camel | Cattle | Dog | Pig | Chicken | Sheep | Duck | ||||
Sheep | 15 | 5 (33.3%) | ||||||||
meat balls | 5 | mutton | 1/5 a, 1/5 b | 1/5 a | 5/5 | 1/5 b | ||||
meat slices | 5 | mutton | 1/5 | 5/5 | ||||||
kebab | 5 | mutton | 2/5 | 5/5 | ||||||
Cattle | 15 | 4 (26.7%) | ||||||||
meat balls | beef | 5/5 | 1/5 a, 1/5 b | 1/5 c | 1/5 a | |||||
meat slices | beef | 5/5 | ||||||||
kebab | beef | 5/5 | 1/5 | |||||||
Camel | 10 | 1 (10%) | ||||||||
drysaltery | 5 | camel | 5/5 | 1/5 | ||||||
jerky | 5 | camel | 5/5 |
Multiplex PCR Type | Species Number | Detection Items | Detection Limit | Detection Method | Reference or Source |
---|---|---|---|---|---|
Heptaplex | 7 | camel, cattle, dog, pig, chicken, sheep and duck | 0.025 ng DNA or 0.1% for each species | Gel | This study |
Multiplex | 3 | chicken, turkey, duck | 1 pg for each species | Gel | [29] |
Tetraplex | 3 | pig, cattle, fish | 0.001–0.1 ng DNA | Gel | [30] |
Multiplex | 4 | ruminant, poultry, pork, donkey | 0.01–0.1 ng/μL DNA | Gel | [1] |
Multiplex | 4 | pork, chicken, duck, cattle | 0.1% for each species | Gel | [31] |
tetraplex | 4 | horse, soybean, poultry, pork | 0.01% for each species | Gel | [32] |
Multiplex | 4 | chicken, duck, pork, beef | 0.05% for each species | Gel | [33] |
Multiplex | 4 | buffalo, cattle, pork, duck | 1 pg DNA, 0.1% for each species | Gel | [34] |
Quadruplex | 4 | chicken, mutton, beef, pork | 16 pg DNA, 0.01% of each species | Gel | [35] |
Multiplex | 4 | rat, fox, duck, sheep | 0.05 ng/μL DNA | Gel | [36] |
Multiplex | 5 | cat, dog, pig, monkey, rat | 0.01–0.02 ng DNA | chip | [19] |
Multiplex | 5 | sheep/goat, bovine, chicken, duck, pig | 0.5 ng DNA | Gel | [26] |
Hexaplex | 6 | horse, soybean, sheep, poultry, pork, cow | 0.01% for each species | Gel | [37] |
Hexaplex | 6 | chicken, cow/buffalo, sheep/goat, horse/donkey, pork, dog | 0.03–0.05 ng DNA | Gel | [25] |
Multiplex | 6 | goat, chicken, cattle, sheep, pig, horse | 0.25 ng DNA | Gel | [38] |
Multiplex | 6 | mutton, pork, duck, chicken, horse, cat | 9.1% of each species | Gel | [39] |
Septuple | 7 | turkey, goose, pig, sheep, beef, chicken, duck | 0.01–0.05 ng DNA | Gel | [17] |
Heptaplex | 7 | pig, beef, horse, duck, chicken, pigeon, camel | 0.01–0.025 ng DNA or 0.1% for each species | Gel | [3] |
Octuplex | 8 | dog, chicken, cattle, pig, horse, donkey, fox, rabbit | 0.05 ng/μL DNA | Gel | [21] |
Multiplex (two-tube) | 10 | beef, sheep, pork, chicken, turkey; cat, dog, mouse, rat, human | 30 pg DNA | Gel | [24] |
Multiplex (two-tube) | 12 | horse, pigeon, camel, rabbit, ostrich, beef; turkey, dog, chicken, duck, cat, goose | 0.05–0.1 ng DNA | Gel | [28] |
Multiplex (two-tube) | 14 | cattle, donkey, canidae (dog, fox, raccoon-dog), deer, horse; pig, ovis (sheep, goat), poultry (chicken, duck), cat, mouse | 0.02–0.2 ng DNA | Chip | [6] |
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Cheng, Y.; Wang, S.; Ju, S.; Zhou, S.; Zeng, X.; Wu, Z.; Pan, D.; Zhong, G.; Cai, Z. Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach. Nutrients 2022, 14, 4727. https://doi.org/10.3390/nu14224727
Cheng Y, Wang S, Ju S, Zhou S, Zeng X, Wu Z, Pan D, Zhong G, Cai Z. Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach. Nutrients. 2022; 14(22):4727. https://doi.org/10.3390/nu14224727
Chicago/Turabian StyleCheng, Yan, Sha Wang, Shilong Ju, Song Zhou, Xiaoqun Zeng, Zhen Wu, Daodong Pan, Guowei Zhong, and Zhendong Cai. 2022. "Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach" Nutrients 14, no. 22: 4727. https://doi.org/10.3390/nu14224727