Development and validation of a SNPs panel used for beef traceability throughout the food chain

LAMAS, Alexandre; BARREIRO, Rocio; REGAL, Patricia; VÁZQUEZ, Beatriz; MIRANDA, José Manuel; CEPEDA, Alberto; FRANCO, Carlos Manuel

doi:10.1590/fst.07221

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Original Article • Food Sci. Technol (Campinas) 42 • 2022 • https://doi.org/10.1590/fst.07221 copy

Development and validation of a SNPs panel used for beef traceability throughout the food chain

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Meat labelling is important to avoid unfair competition practices of producers, processors and sellers aiming gain an economic advantage from misrepresenting cheaper meat products as premium quality ones. Genetic traceability is an ideal tool for the detection and prevention of fraud in the sale of beef. In this work, a method for validating protected status based on a combination of single nucleotide polymorphisms (SNPs) and a high-throughput real-time PCR protocol was developed. A total of 1,911 beef samples were used to develop and validate an OpenArray® panel consisting of 26 SNPs selected from an ISAG-ICAR panel. The applicability of the method was determined by comparing 143 blind pairs of samples. The combined probability of identity (PI) with the 26 SNPs selected was 2.09-11 with 22 SNPs showing an individual PI lower than 0.4. In all tests, the blind pair of samples were correctly as-signed. The panel designed and validated in this study is of great utility to confirm genetic content of the specific genetic group tested and detect fraud in a routine way that requires a minimum amount of time and is cost effective.

Keywords:
SNPs; fraud; beef; traceability; real-time PCR; OpenArray®

Chromosome	Accession Nº NCBI	Locus identifier	Assay ID	Primer and probe sequences	Context Sequence	Alelles
18	AY914316.1	BTA18 3144	AHQJU6T	For GAGGAGAGCTCTTCTCAGATTGC	ATGAAAGGAAT[A/C]GGAAATC	A,C
				Rev TGCTCTCCTTGTCTGGGAAGA
				VIC ATGAAAGGAATAGGAAATC
				FAM AAAGGAATCGGAAATC
1	DQ404149.1	BTA01 BES6	AHI155I	For GGGCTCATAAGATAAAGCTTTTGCT	TCAGCATTTCTT[T/C]GAGATTA	T,C
				Rev GGGCATAGAACTCCTTGAAAATGG
				VIC TCAGCATTTCTTTGAGATTA
				FAM AGCATTTCTTCGAGATTA
16	DQ846693.1	BTA16 349064_338	AHS1RI9	For AGGGTTTTCAGGCTTTATGTCTGT	TTTGCCTATG[A/G]TTGCCTG	A,G
				Rev AGACCAGCTTCTTGGCTTCTG
				VIC TTTGCCTATGATTGCCTG
				FAM TGCCTATGGTTGCCTG
28	EF042091.1	BTA28 186736_5402	AHUAPPH	For TGATGAAGCAATAAGGCACTGATGA	CCCAGCCCTT[T/C]ACCACA	T,C
				Rev GGTAAACTCATCACCTCCAAACTTTTG
				VIC CCCAGCCCTTTACCACA
				FAM CCAGCCCTTCACCACA
3	DQ489377.1	BTA03 240852_12041	AHVJNVP	For CTCTGTACAGCAGGAGAAATAGCAA	CTGCATCA[A/G]ACAAACC	A,G
				Rev TGTGCCGACCTCACGTG
				VIC CTGCATCAAACAAACC
				FAM TGCATCAGACAAACC
23	AY937242.1	BTA23 5279	AHWSL1X	For AAAACAGAGCAAAAATCCTTGCCTT	CCCATTCCCTA[T/C]CCCATG	T,C
				Rev ACACAAAAAATACATTTAGTTATTTATTATGTATATTGTCTGTCT
				VIC CCCATTCCCTATCCCATG
				FAM CCATTCCCTACCCCATG
11	AY851162.1	BTA11 IL1B	AH0JGKL	For CTCTCTCCCTAAAGAAAGCCATACC	TGAATAACCCC[A/G]AGGACTG	A,G
				Rev GCTACATTCTTCCCTTCCCTTCTG
				VIC TGAATAACCCCAAGGACTG
				FAM AATAACCCCGAGGACTG
1	DQ381153.1	BTA01 135_21121	AH1SEQT	For CTCCCAAGGGCATCTCTCTAC	CCAGGCATTA[A/C]TATTAC	A,C
				Rev TGTCCTTTGTCTGTCAGAATGGAT
				VIC CCAGGCATTAATATTAC
				FAM CCAGGCATTACTATTAC
5	DQ500958	BTA05 BES11	AH21CW1	For GCACTCCCTTCCCAGAACAG	CTCTGCTAATG[T/C]TTTCAG	T,C
				Rev ACCCAGACAAAGGTCAATGCTT
				VIC CTCTGCTAATGTTTTCAG
				FAM TCTGCTAATGCTTTCAG
8	DQ916058.1	BTA08 302326_838	AH4AA29	For ACCAGGGCATTACCTATCACAGA	TGTCCGTTTCC[T/C]TTAAAAT	T,C
				Rev GGTTAGACTTGGCGCTCTTAATAGA
				VIC TGTCCGTTTCCTTTAAAAT
				FAM TCCGTTTCCCTTAAAAT
18	EF028073.1	BTA18 186240_12002	AH5I89H	For CAAGATGATAAGGATTGTATTTAAGGTTGTCT	TCGCATTAATTAGT[A/G]TCTACC	A,G
				Rev GTCCTGCCTGTCAATAATCAAGTTG
				VIC TCGCATTAATTAGTATCTACC
				FAM CGCATTAATTAGTGTCTACC
6	DQ647190.1	BTA06 106320_3036	AH6R7FP	For GTCCAGGGACACCAGTCTATG	CTCCATGTC[A/G]TTGGAAC	A,G
				Rev CCCAAGTACGAAAGGGTTAGTGAT
				VIC CTCCATGTCATTGGAAC
				FAM TCCATGTCGTTGGAAC
29	EF034080.1	BTA29 50303_4748	AH705LX	For CACCACGGTCACATGATCCT	CAATATGGC[A/G]GTGCTTC	A,G
				Rev AGCTATTAATACGTATTCCATTCTATAGTGT
				VIC CAATATGGCAGTGCTTC
				FAM AATATGGCGGTGCTTC
22	EF093509.1	BTA22 190836_5512	AHABIXY	For CTGTGTGATTGGCCAAAAGGTATT	CAAACAATACTAAAATC[A/T]ATTATC	A,T
				Rev TGGTGTCCTTAGCAAATAAATGAATGAATAAAT
				VIC AACAATACTAAAATCAATTATC
				FAM CAAACAATACTAAAATCTATTATC
12	DQ786763.1	BTA12 215881_4945	AHBKG36	For AGAAGCTTCTGGCCCAGAGA	TGTTTTAGCAAAC[T/A]ACATTTG	T,A
				Rev GTCTTTTCCTGAGTCCCCACATTAT
				VIC TGTTTTAGCAAACTACATTTG
				FAM TTAGCAAACAACATTTG
25	EF034083.1	BTA25 60020_10226	AHCTFAE	For CGGAGGTTTGGAGTCTTGTTTTCT	AGTCAATCTTCA[A/C]GCAAAAT	A,C
				Rev GCCAGCACCTCCCATTCTAT
				VIC AGTCAATCTTCAAGCAAAAT
				FAM CAATCTTCACGCAAAAT
29	DQ404153.1	BTA29 11629	AHD2DGM	For TCCTGCAGTTCTCTCCTGTCT	TCAGGGCTC[A/G]GTGAGC	A,G
				Rev GGACCATGTGGCCAAGCA
				VIC TCAGGGCTCAGTGAGC
				FAM AGGGCTCGGTGAGC
6	DQ789028.1	BTA06 35310_9826	AHFBBMU	For TCAAAGCAACACATCTGGTCATCA	CAACCTGCT[T/C]GGCTCT	T,C
				Rev AATTAACTCTCTCAAATCTGAGTGAAGTG
				VIC CAACCTGCTTGGCTCT
				FAM AACCTGCTCGGCTCT
15	DQ866817.1	BTA15 BES7	AHGJ9S2	For GTCCAGCATCTTTAACCCTGACTAA	CAATCTACT[A/G]AAATTCC	A,G
				Rev GTGGTGATCTCAAAGAGGCTTTCA
				VIC CAATCTACTAAAATTCC
				FAM AATCTACTGAAATTCC
26	AY943841	BTA26 12063	AHHS7ZA	For ACTCTAGAGGGCCTAAACTCCAAAT	CTTCTGTCCC[A/G]AGTCAAG	A,G
				Rev GGCCTGCTTCTCACAAAGACA
				VIC CTTCTGTCCCAAGTCAAG
				FAM TCTGTCCCGAGTCAAG
4	DQ647186.1	BTA04 116028_6113	AHKA4BQ	For TGGCTAGGGATCTGACTGCTT	CCTCACAGCT[A/G]TGTCTTT	A,G
				Rev GAAGACAGGGAGAAGGGAAAGTTTA
				VIC CCTCACAGCTATGTCTTT
				FAM TCACAGCTGTGTCTTT
19	AY916666.1	BTA19 5067	AHLJ2HY	For ATCTGAGAGGTGGGACAAGGA	TGCGGC[T/C]AAGCCAC	T,C
				Rev CCTTTCCCAGAGACTCAGACTTG
				VIC TGCGGCTAAGCCAC
				FAM CGGCCAAGCCAC
26	EF150946.1	BTA26 90532_852	AHZAIED	For CACTTCTCTCTCTGCTTTCCCA	CCACCGC[A/G]TGGTTAT	A,G
				Rev TTCCAGAAGGGCACCAAGAAAA
				FAM CCACCGCATGGTTAT
				VIC CACCGCGTGGTTAT
5	AY844963.1	BTA05 16075	AHX1J75	For TCTTTGTATGTTTTAAATCTTGTTTTTCCTTCTGT	CCCTTTTGTACA[T/C]TGGTTAC	T,C
				Rev TCAGATATCCTGAAGGTGAGATCCAA
				VIC CCCTTTTGTACATTGGTTAC
				FAM CCTTTTGTACACTGGTTAC
20	DQ888313.1	BTA20 BES1	AH893R5	For CTGGCAGAGTTCGGGTTTAGT	CACCATGCCA[T/C]GTAAAT	T,C
				Rev GCTTCTGGAGCTCACCCA
				VIC CACCATGCCATGTAAAT
				FAM CCATGCCACGTAAAT
10	DQ786761.1	BTA10 135062_12754	AHRSTC1	For CATATACCATAGTATTTTTTCCCTTAGCTTACTGA	TGCTTTTTATAAG[A/G]TTTAAC	A,G
				Rev CTAGACCCTTTTCAAGTTTCTTTTGTTTCTT
				VIC TGCTTTTTATAAGATTTAAC
				FAM TGCTTTTTATAAGGTTTAAC

		Allele frequencies		Genotype frequencies observed
Assay	UND/NOAMP*	a	b	a,a	a,b	b,b	P_I
AH0JGKL	86 (4.5%)	0.28	0.72	0.08	0.40	0.52	0.438
AH1SEQT	78 (4.08%)	0.50	0.50	0.25	0.51	0.24	0.381
AH21CW1	100 (5.23%)	0.47	0.53	0.23	0.47	0.29	0.364
AH4AA29	108 (5.65%)	0.50	0.50	0.26	0.48	0.26	0.365
AH5I89H	95 (4.97%)	0.51	0.49	0.25	0.50	0.24	0.378
AH6R7FP	90 (4.7%)	0.40	0.60	0.17	0.47	0.37	0.381
AH705LX	90 (4.7%)	0.70	0.30	0.50	0.40	0.10	0.419
AH893R5	87 (4.55%)	0.60	0.40	0.36	0.50	0.15	0.394
AHABIXY	288 (15.00%)	0.55	0.45	0.31	0.47	0.22	0.367
AHBKG36	112 (5.86%)	0.41	0.59	0.04	0.74	0.22	0.600
AHCTFAE	87 (4.55%)	0.39	0.61	0.15	0.48	0.37	0.389
AHD2DGM	58 (3.03%)	0.54	0.46	0.30	0.48	0.22	0.370
AHFBBMU	78 (4.08%)	0.61	0.39	0.37	0.48	0.15	0.387
AHGJ9S2	45 (2.35%)	0.49	0.51	0.24	0.49	0.27	0.370
AHHS7ZA	26 (1.36%)	0.50	0.50	0.24	0.51	0.25	0.379
AHI155I	105 (5.49%)	0.49	0.51	0.24	0.51	0.26	0.379
AHKA4BQ	92 (4.81%)	0.51	0.49	0.26	0.50	0.24	0.376
AHLJ2HY	84 (4.39%)	0.35	0.65	0.13	0.44	0.43	0.396
AHQJU6T	87 (4.55%)	0.47	0.53	0.24	0.47	0.29	0.364
AHRSTC1	36 (1.88%)	0.47	0.53	0.25	0.45	0.31	0.355
AHS1RI9	21 (1.09%)	0.66	0.34	0.43	0.47	0.10	0.414
AHUAPPH	92 (4.81%)	0.46	0.54	0.20	0.52	0.28	0.389
AHVJNVP	115 (6.01%)	0.55	0.45	0.29	0.52	0.19	0.388
AHWSL1X	35 (1.83%)	0.59	0.41	0.36	0.47	0.18	0.377
AHX1J75	101 (5.28%)	0.47	0.53	0.22	0.49	0.28	0.374
AHZAIED	27 (1.41%)	0.55	0.45	0.33	0.45	0.22	0.359
Combined							2.09E-11

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[1] *Corresponding Author: alexandre.lamas@usc.es