Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations
Abstract
:1. Introduction
2. Materials and Methods
2.1. Overall Description of the Stepwise Approach
2.2. Confirmatory Sequencing Analysis of CRISPR-Cas9 Mutations
2.3. Amplicon Sequence Search for Natural Variants
2.4. RT-PCR Primer and Probe Design
2.5. In Silico PCR Testing
2.6. RT-qPCR Empirical Assay
3. Results
3.1. Sequence Confirmation of Mutated Alleles
3.2. In Silico Specificity Assessment
3.3. In Silico PCR Performance
3.4. Real-Time qPCR Performance and Empirical Primer Specificity Evaluation
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Sequence Number | Hits | Accession | BLAST against the Query grf1-3 | (MSA) Mismatches (bp) or Gaps against Each Accession | |||
---|---|---|---|---|---|---|---|
Query Cover | Per. Ident | Forward | Probe | Reverse | |||
1 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR782543.1 | 100% | 99.24% | 1 | 0 | 0 |
2 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699746.2 | 100% | 99.24% | 1 | 0 | 0 |
3 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699771.1 | 100% | 99.24% | 1 | 0 | 0 |
4 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699766.1 | 100% | 99.24% | 1 | 0 | 0 |
5 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699761.1 | 100% | 99.24% | 1 | 0 | 0 |
6 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699756.1 | 100% | 99.24% | 1 | 0 | 0 |
7 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR699751.1 | 100% | 99.24% | 1 | 0 | 0 |
8 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR215053.1 | 100% | 99.24% | 1 | 0 | 0 |
9 | Arabidopsis thaliana growth-regulating factor 1 (GRF1), mRNA | NM_127849.4 | 100% | 99.24% | 1 | 0 | 0 |
10 | Arabidopsis thaliana chromosome 2 | CP116281.1 | 100% | 99.24% | 1 | 0 | 0 |
11 | Arabidopsis thaliana genome assembly, chromosome: 2 | OX298798.1 | 100% | 99.24% | 1 | 0 | 0 |
12 | Arabidopsis thaliana genome assembly, chromosome: 2 | OX298803.1 | 100% | 99.24% | 1 | 0 | 0 |
13 | Arabidopsis thaliana ecotype 1254 chromosome 2 sequence | CP086755.1 | 100% | 99.24% | 1 | 0 | 0 |
14 | Arabidopsis thaliana ecotype 5856 chromosome 2 sequence | CP086750.1 | 100% | 99.24% | 1 | 0 | 0 |
15 | Arabidopsis thaliana ecotype 6021 chromosome 2 sequence | CP086745.1 | 100% | 99.24% | 1 | 0 | 0 |
16 | Arabidopsis thaliana ecotype 6024 chromosome 2 sequence | CP086740.1 | 100% | 99.24% | 1 | 0 | 0 |
17 | Arabidopsis thaliana ecotype 9412 chromosome 2 sequence | CP086735.1 | 100% | 99.24% | 1 | 0 | 0 |
18 | Arabidopsis thaliana ecotype 9470 chromosome 2 sequence | CP086730.1 | 100% | 99.24% | 1 | 0 | 0 |
19 | Arabidopsis thaliana chromosome 2 | CP087127.2 | 100% | 99.24% | 1 | 0 | 0 |
20 | Arabidopsis thaliana isolate t2t_salk_col chromosome 2 | CP096025.1 | 100% | 99.24% | 1 | 0 | 0 |
21 | Arabidopsis thaliana genome assembly, chromosome: 2 | OW119597.1 | 100% | 99.24% | 1 | 0 | 0 |
22 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR881467.1 | 100% | 99.24% | 1 | 0 | 0 |
23 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR797808.1 | 100% | 99.24% | 1 | 0 | 0 |
24 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR797803.1 | 100% | 99.24% | 1 | 0 | 0 |
25 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR797798.1 | 100% | 99.24% | 1 | 0 | 0 |
26 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR797793.1 | 100% | 99.24% | 1 | 0 | 0 |
27 | Arabidopsis thaliana genome assembly, chromosome: 2 | LR797788.1 | 100% | 99.24% | 1 | 0 | 0 |
28 | Arabidopsis thaliana chromosome 2 | CP002685.1 | 100% | 99.24% | 1 | 0 | 0 |
29 | Arabidopsis thaliana At2g22840 mRNA for hypothetical protein, partial cds, clone: RAAt2g22840 | AB493560.1 | 100% | 99.24% | 1 | 0 | 0 |
30 | Arabidopsis thaliana isolate CS906 GRL1 (GRL1) gene, partial cds | EU550462.1 | 100% | 99.24% | 1 | 0 | 0 |
31 | Arabidopsis thaliana isolate CS902 GRL1 (GRL1) gene, partial cds | EU550456.1 | 100% | 99.24% | 1 | 0 | 0 |
32 | Arabidopsis thaliana isolate CS6799 GRL1 (GRL1) gene, partial cds | EU550455.1 | 100% | 99.24% | 1 | 0 | 0 |
33 | Arabidopsis thaliana isolate CS901 GRL1 (GRL1) gene, partial cds | EU550445.1 | 100% | 99.24% | 1 | 0 | 0 |
34 | Arabidopsis thaliana transcription activator (GRF1) mRNA, complete cds | AY102634.1 | 100% | 99.24% | 1 | 0 | 0 |
35 | Arabidopsis thaliana chromosome 2 clone T20K9 map CIC06C07, complete sequence | AC004786.3 | 100% | 99.24% | 1 | 0 | 0 |
36 | Arabidopsis thaliana Full-length cDNA Complete sequence from clone GSLTPGH12ZD08 of Hormone-Treated Callus of strain col-0 of Arabidopsis thaliana (thale cress) | BX820248.1 | 100% | 99.24% | 1 | 0 | 0 |
37 | PREDICTED: Arabidopsis xampl subsp. xampl growth-regulating factor 1 (LOC9316532), mRNA | XM_002878592.2 | 100% | 98.47% | 1 | 0 | 0 |
38 | PREDICTED: Camelina sativa growth-regulating factor 1-like (LOC104713726), mRNA | XM_010430916.2 | 100% | 98.47% | 1 | 0 | 0 |
39 | PREDICTED: Camelina sativa growth-regulating factor 1 (LOC104751923), mRNA | XM_010473979.2 | 100% | 98.47% | 1 | 0 | 0 |
40 | PREDICTED: Camelina sativa growth-regulating factor 1-like (LOC104704976), mRNA | XM_010420970.1 | 100% | 98.47% | 1 | 0 | 0 |
41 | Camelina hispida cultivar hispida voucher DAO 902780 chromosome 2 | CP094632.1 | 100% | 97.71% | 1 | 0 | 1 |
42 | Arabidopsis arenosa genome assembly, chromosome: 4 | LR999454.1 | 100% | 97.71% | 1 | 0 | 1 |
43 | Raphanus sativus genome assembly, chromosome: 6 | LR778315.1 | 98% | 96.12% | 1 | 0 | 2 (gap) |
44 | PREDICTED: Raphanus sativus growth-regulating factor 1 (LOC108836427), mRNA | XM_018609585.1 | 98% | 96.12% | 1 | 0 | 2 (gap) |
45 | PREDICTED: Brassica rapa growth-regulating factor 1 (LOC103858395), mRNA | XM_009135745.3 | 100% | 95.42% | 1 | 0 | 1 |
46 | Brassica oleracea HDEM genome, scaffold: C3 | LR031872.1 | 100% | 95.42% | 1 | 0 | 1 |
47 | Brassica rapa genome, scaffold: A03 | LR031572.1 | 100% | 95.42% | 1 | 0 | 1 |
48 | PREDICTED: Capsella rubella growth-regulating factor 1 (LOC17887921), mRNA | XM_006293922.2 | 100% | 95.42% | 1 | 1 | 1 |
49 | PREDICTED: Brassica napus growth-regulating factor 1-like (LOC125584397), mRNA | XM_048752816.1 | 100% | 95.42% | 1 | 0 | 1 |
50 | PREDICTED: Brassica napus growth-regulating factor 1 (LOC106389497), mRNA | XM_013829762.3 | 100% | 95.42% | 1 | 0 | 1 |
51 | Brassica rapa genome assembly, chromosome: A03 | LS974619.2 | 100% | 95.42% | 1 | 0 | 1 |
52 | Brassica napus genome assembly, chromosome: C03 | HG994367.1 | 100% | 95.42% | 1 | 0 | 1 |
53 | Brassica napus genome assembly, chromosome: A03 | HG994357.1 | 100% | 95.42% | 1 | 0 | 1 |
54 | Brassica rapa subsp. Pekinensis growth-regulating xamp 1 mRNA, partial cds | JN698986.1 | 100% | 95.42% | 1 | 0 | 1 |
55 | PREDICTED: Brassica oleracea var. oleracea growth-regulating factor 1 (LOC106328366), mRNA | XM_013766798.1 | 100% | 94.66% | 1 | 0 | 1 |
56 | PREDICTED: Eutrema salsugineum growth-regulating factor 1 (LOC18021800), mRNA | XM_006404687.2 | 100% | 93.89% | 1 | 2 | 2 |
57 | Arabis alpina genome assembly, chromosome: 6 | LT669793.1 | 93% | 95.90% | 1 | 2 | 9 (gap) |
Sequence Number | Description | Accession | BLAST against the Query grf8-61 | (MSA) Mismatches (bp) or Gaps against Each Accession | |||
---|---|---|---|---|---|---|---|
Query Cover | Per. Ident | Forward | Probe | Reverse | |||
1 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR782545.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
2 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699748.2 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
3 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699773.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
4 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699768.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
5 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699758.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
6 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699753.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
7 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR215055.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
8 | Arabidopsis thaliana chromosome 4 | CP116283.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
9 | Arabidopsis thaliana genome assembly, chromosome: 4 | OX298800.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
10 | Arabidopsis thaliana genome assembly, chromosome: 4 | OX298805.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
11 | Arabidopsis thaliana ecotype 1254 chromosome 4 sequence | CP086757.1 | 98% | 99.20% | 0 | 0 | 4 (gaps) 1 mismatch |
12 | Arabidopsis thaliana ecotype 5856 chromosome 4 sequence | CP086752.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
13 | Arabidopsis thaliana ecotype 6021 chromosome 4 sequence | CP086747.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
14 | Arabidopsis thaliana ecotype 6024 chromosome 4 sequence | CP086742.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
15 | Arabidopsis thaliana ecotype 9412 chromosome 4 sequence | CP086737.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
16 | Arabidopsis thaliana ecotype 9470 chromosome 4 sequence | CP086732.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
17 | Arabidopsis thaliana chromosome 4 | CP087129.2 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
18 | Arabidopsis thaliana isolate t2t_salk_col chromosome 4 | CP096027.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
19 | Arabidopsis thaliana genome assembly, chromosome: 4 | OW119599.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
20 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR881469.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
21 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR797810.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
22 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR797805.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
23 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR797800.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
24 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR797795.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
25 | Arabidopsis thaliana chromosome 4 | CP002687.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
26 | Arabidopsis thaliana DNA chromosome 4, contig xample No. 61 | AL161561.2 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
27 | Arabidopsis thaliana DNA chromosome 4, BAC clone T19F6, partial sequence (ESSA xample) | AL109619.1 | 96% | 100.00% | 0 | 0 | 5 (gaps) |
28 | Arabidopsis thaliana chromosome IV BAC T19F6 genomic sequence, complete sequence | AC002343.1 | 98% | 99.20% | 0 | 0 | 4 (gaps) 1 mismatch |
29 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR699763.1 | 96% | 97.54% | 0 | 2 | 5 (gaps) |
30 | Arabidopsis thaliana genome assembly, chromosome: 4 | LR797790.1 | 96% | 97.54% | 0 | 2 | 5 (gaps) |
31 | Arabidopsis thaliana growth-regulating factor 8 (GRF8), partial mRNA | NM_118547.2 | 83% | 100.00% |
Total of Mismatches | Number/Hits Analyzed | Sequences Corresponding Perfectly to the Primer | Number of BLAST Hits Recovered | Possible Discrimination between the grf1-3 Genotype and Other Lines |
---|---|---|---|---|
1 | 39 | 0 | 57 | Low |
2 | 17 | 0 | ||
3 | 1 | 0 |
Total of Mismatches | Number/Hits Analyzed | Sequences Corresponding Perfectly to the Primers | Number of BLAST Hits Recovered | Possible Discrimination between the grf8-3 Genotype and Other Lines |
---|---|---|---|---|
1 | 1 | 1 | 1 | High |
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Zanatta, C.B.; Hoepers, A.M.; Nodari, R.O.; Agapito-Tenfen, S.Z. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods 2023, 12, 4298. https://doi.org/10.3390/foods12234298
Zanatta CB, Hoepers AM, Nodari RO, Agapito-Tenfen SZ. Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations. Foods. 2023; 12(23):4298. https://doi.org/10.3390/foods12234298
Chicago/Turabian StyleZanatta, Caroline Bedin, Aline Martins Hoepers, Rubens Onofre Nodari, and Sarah Zanon Agapito-Tenfen. 2023. "Specificity Testing for NGT PCR-Based Detection Methods in the Context of the EU GMO Regulations" Foods 12, no. 23: 4298. https://doi.org/10.3390/foods12234298