转基因大豆多靶标质粒DNA标准分子的研制及应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0128

摘要/Abstract

摘要：

【目的】 为了确保转基因检测技术的标准化和准确性，开发一种适用于转基因大豆定性定量检测的多靶标质粒标准分子。【方法】 针对13种转基因大豆转化体的分子特征信息，合成了含有多靶标序列的核酸片段，通过将外源片段插入pUC57质粒多克隆位点并经大肠杆菌转化，研制了含有27个转基因大豆靶标序列的质粒DNA标准分子pUC57-SOY。采用普通PCR、单重、双重实时荧光定量PCR（qPCR）检测方法对DNA标准分子pUC57-SOY的靶标特异性进行测试，利用13种大豆转化体进行定量检测适用范围和定值参数性能进行评估。【结果】 在普通PCR检测中，质粒DNA包含的24种检测靶标，均能稳定扩增，且无非特异性扩增产物，表明质粒DNA标准分子pUC57-SOY在实际检测中具有良好的特异性；实时荧光定量PCR（qPCR）方法测试表明，质粒标准分子的不同浓度与靶标扩增效率具有良好的线性相关性。以pUC57-SOY标准分子为阳性参照物，应用标准曲线内插法，实际样品检测结果与预期相一致。【结论】 开发的多靶标质粒标准分子pUC57-SOY表现出符合转基因成分定性定量检测要求的优良性能。在定性检测中，24种检测靶标均能稳定检测出并具有良好的特异性；在定量检测中，可实现一个标准分子对应13种靶标的精准测定。因此，该研究创制的质粒标准分子pUC57-SOY可作为转基因大豆检测的阳性对照品。

关键词: 转基因成分检测, 转基因大豆, 质粒DNA标准分子, 定量

Abstract:

【Objective】 In order to ensure the standardization and accuracy of transgenic detection technology, this study is aimed to develop a multi-target plasmid standard molecule suitable for qualitative and quantitative detection of transgenic soybean. 【Method】 In order to characterize molecular features of 13 transgenic soybean events, nucleic acid fragments containing multi-target sequences were synthesized. These fragments were inserted into the multiple cloning sites of the pUC57 plasmid and transformed into Escherichia coli, resulting in the development of a plasmid DNA standard molecule, pUC57-SOY, containing 27 transgenic soybean target sequences. The target specificity of the DNA standard molecule pUC57-SOY was tested using conventional PCR, single, and duplex real-time fluorescence quantitative PCR（qPCR）detection methods. The applicability range and quantitative parameters were evaluated using 13 transgenic soybean events for quantitative detection. 【Result】 In PCR detection, all 24 target sequences contained in the plasmid DNA were consistently amplified without non-specific amplification products, indicating the excellent specificity of the plasmid DNA standard molecule pUC57-SOY in practical detection. Real-time fluorescence quantitative PCR（qPCR）analysis demonstrated a good linear correlation between different concentrations of the plasmid standard molecule and target amplification efficiency. Using pUC57-SOY as a positive reference, the actual sample detection results were consistent with expectations when applying the standard curve interpolation method. 【Conclusion】 The multi-target plasmid standard molecule pUC57-SOY developed in this study showed excellent performance in line with the requirements of qualitative and quantitative detection of transgenic components. In qualitative detection, 24 detection targets can be stably detected and have good specificity. In quantitative detection, a standard substance corresponding to 13 kinds of targets can be accurately determined. Therefore, the plasmid standard molecule pUC57-SOY created in this study can be used as a positive control product for the detection of transgenic soybeans.

Key words: detection of transgenic composition, transgenic soybean, plasmid DNA standard molecule, quantifative

刘奕君, 闫伟, 何禹璇, 董立明, 龙丽坤, 李飞武. 转基因大豆多靶标质粒DNA标准分子的研制及应用[J]. 生物技术通报, 2024, 40(11): 169-183.

LIU Yi-jun, YAN Wei, HE Yu-xuan, DONG Li-ming, LONG Li-kun, LI Fei-wu. Development and Application of DNA Standard Molecules of Transgenic Soybean Multi-target Plasmid[J]. Biotechnology Bulletin, 2024, 40(11): 169-183.

图/表 10

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序号 Serial number	检测靶标 Detection target	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	预期产物大小Expected product size/bp
1	lectin	lec-F	GCCCTCTACTCCACCCCCATCC	lec-R	GCCCATCTGCAAGCCTTTTTGTG	118
2	P-CaMV35S	P-35S-F	GCTCCTACAAATGCCATCATTGC	P-35S-R	GATAGTGGGATTGTGCGTCATCCC	195
3	T-CaMV35S	T35S-F	GTTTCGCTCATGTGTTGAGC	T35S-R	GGGGATCTGGATTTTAGTACTG	121
4	T-NOS	T-NOS-F	GAATCCTGTTGCCGGTCTTG	T-NOS-R	TTATCCTAGTTTGCGCGCTA	180
5	P-FMV35S	FMV35S-F	AAGACATCCACCGAAGACTTA	FMV35S-R	AGGACAGCTCTTTTCCACGTT	210
6	T-E9-3'	E9 3'-F	TTATGGCATTGGGAAAACTGT	E9 3'-R	GGTCATTAGAGGCCACGATT	273
7	NptII	npt II-F	ACTGGGCACAACAGACAATCG	npt II-R	GCATCAGCCATGATGGATACTTT	289
8	bar	bar-F	GAAGGCACGCAACGCCTACGA	bar-F	CCAGAAACCCACGTCATGCCA	262
9	pat	pat-F	GAAGGCTAGGAACGCTTACGA	pat-R	CCAAAAACCAACATCATGCCA	262
10	cry1Ac	cry1Ac-F	ACCGGTTACACTCCCATCGACATC	cry1Ac-R	GGCGGAGTTCATGTCGTTGAA	330
11	CP4-epsps	CP4-F	ACGGTGAYCGTCTTCCMGTTAC	CP4-R	GAACAAGCARGGCMGCAACCA	333
12	MON87708	87708-F	CCATCATACTCATTGCTGATCCA	87708-R	AGCCAATCAATCTCAGAACTGTC	233
13	MON87701	87701-F	GCACGCTTAGTGTGTGTGTCAAAC	87701-R	GGATCCGTCGACCTGCAGTTAAC	150
14	MON87705	87705-F	CGCCAAATCGTGAAGTTTCTCATCT	87705-R	CAGTGATAACAACACCCTGAGTCT	318
15	MON87769	87769-F	CCGGACATGAAGCCATTTAC	87769-R	TCCTTGGAGGTCGTCTCATT	298
16	A2704^[13]	A2704-F	TGAGGGGGTCAAAGACCAAG	A2704-R	CCAGTCTTTACGGCGAGT	239
17	DAS68416	68416-F	CCGCTACTTGCTCTTGTCGT	68416-R	CGGTTAGGATCCGGTGAGTA	221
18	CV127^[14]	CV127-F	CCTTCGCCGTTTAGTGTATAGG	CV127-R	AGCAGGTTCGTTTAAGGATGAA	238
19	GTS40-3-2^[15]	GTS40-F	TTCAAACCCTTCAATTTAACCGAT	GTS40-R	AAGGATAGTGGGATTGTGCGTC	370
20	A5547^[16]	A5547-F	TGAGGGGGTCAAAGACCAAG	A5547-R	CCAGTCTTTACGGCGAGT	317
21	356043^[17]	356043-F	CTTTTGCCCGAGGTCGTTAG	356043-R	GCCCTTTGGTCTTCTGAGACTG	145
22	FG72	FG72-F	TCGGGCTGCAGGAATTAATGT	FG72-R	TTTGGAGCAATAAACATGTGATAGC	150
23	MON89788^[18]	89788-F	CTGCTCCACTCTTCCTTT	89788-R	AGACTCTGTACCCTGACCT	223
24	305423^[19]	305423-F	CGTCAGGAATAAAGGAAGTACAGTA	305423-R	GCCCTAAAGGATGCGTATAGAGT	235
25	zSSIIb	zSSIIb-F	CGGTGGATGCTAAGGCTGATG	zSSIIb-R	AAAGGGCCAGGTTCATTATCCTC	88
26	SPS	SPS-F	TTGCGCCTGAACGGATAT	SPS-R	GGAGAAGCACTGGACGAGG	277
27	HMG	HMG-F	TCCTTCCGTTTCCTCGCC	HMG-R	TTCCACGCCCTCTCCGCT	206
28	Sad1	Sad1-F	TGGCCTCTAATCATTGTTATGATG	Sad1-R	TTGAGGTGAGTCAGAATGTTGTTC	282
29	MON810^[20]	MON810-F	CAAGTGTGCCCACCACAGC	MON810-R	GCAAGCAAATTCGGAAATGAA	106
30	MON863^[21]	MON863-F	GCACTCAAAGACCTGGCGAATGA	MON863-R	CCATCTTTGGGACCACTGTCG	411
31	Bt11^[22]	Bt11-F	GCGGAACCCCTATTTGTTTA	Bt11-R	CAAGAAATGGTCTCCACCAAA	93
32	MON89034^[23]	89034-F	GCTGCTACTACTATCAAGCCAATA	89034-R	TGCTTTCGCCTATAAATACGAC	207
33	MIR604^[24]	MIR604-F	TCGCGCGCGGTGTCATCTATG	MIR604-R	CGCGACACACCTCGTTAGTTAA	142
34	TT51^[25]	TT51-F	AGCAGAACTTTAACCCCCGAA	TT51-R	AGAGCCTCGTTGGATTTCTTACAT	274
35	KF6-1	KF6-1-F	CGACAAAAGATCAGGATTTGGG	KF6-1-R	GTTTGGAGTTTAAGAGCGA	303
36	MON1445^[26]	MON1445-F	AATGCTGGATTTTCTGCCTGTG	MON1445-R	TCCAAAAGTCATGCATCATTTCTCA	99
37	MON15985^[27]	MON15985-F	ATTTGATGCACTATGTCTTCGTCTA	MON15985-R	CAATGGAATCCGAGGAGGT	175
38	GT73^[28]	GT73-F	AATAACGCTGCGGACATCTA	GT73-R	CAGCAACATTCTCTGTCAACAA	204
39	T45^[29]	T45-F	TCCCATTTATTTACGGTCAC	T45-R	CCATGGGAATTCATTTACAA	233

序号 Serial number	检测靶标 Detection target	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	预期产物大小Expected product size/bp
1	lectin	lec-F	GCCCTCTACTCCACCCCCATCC	lec-R	GCCCATCTGCAAGCCTTTTTGTG	118
2	P-CaMV35S	P-35S-F	GCTCCTACAAATGCCATCATTGC	P-35S-R	GATAGTGGGATTGTGCGTCATCCC	195
3	T-CaMV35S	T35S-F	GTTTCGCTCATGTGTTGAGC	T35S-R	GGGGATCTGGATTTTAGTACTG	121
4	T-NOS	T-NOS-F	GAATCCTGTTGCCGGTCTTG	T-NOS-R	TTATCCTAGTTTGCGCGCTA	180
5	P-FMV35S	FMV35S-F	AAGACATCCACCGAAGACTTA	FMV35S-R	AGGACAGCTCTTTTCCACGTT	210
6	T-E9-3'	E9 3'-F	TTATGGCATTGGGAAAACTGT	E9 3'-R	GGTCATTAGAGGCCACGATT	273
7	NptII	npt II-F	ACTGGGCACAACAGACAATCG	npt II-R	GCATCAGCCATGATGGATACTTT	289
8	bar	bar-F	GAAGGCACGCAACGCCTACGA	bar-F	CCAGAAACCCACGTCATGCCA	262
9	pat	pat-F	GAAGGCTAGGAACGCTTACGA	pat-R	CCAAAAACCAACATCATGCCA	262
10	cry1Ac	cry1Ac-F	ACCGGTTACACTCCCATCGACATC	cry1Ac-R	GGCGGAGTTCATGTCGTTGAA	330
11	CP4-epsps	CP4-F	ACGGTGAYCGTCTTCCMGTTAC	CP4-R	GAACAAGCARGGCMGCAACCA	333
12	MON87708	87708-F	CCATCATACTCATTGCTGATCCA	87708-R	AGCCAATCAATCTCAGAACTGTC	233
13	MON87701	87701-F	GCACGCTTAGTGTGTGTGTCAAAC	87701-R	GGATCCGTCGACCTGCAGTTAAC	150
14	MON87705	87705-F	CGCCAAATCGTGAAGTTTCTCATCT	87705-R	CAGTGATAACAACACCCTGAGTCT	318
15	MON87769	87769-F	CCGGACATGAAGCCATTTAC	87769-R	TCCTTGGAGGTCGTCTCATT	298
16	A2704^[13]	A2704-F	TGAGGGGGTCAAAGACCAAG	A2704-R	CCAGTCTTTACGGCGAGT	239
17	DAS68416	68416-F	CCGCTACTTGCTCTTGTCGT	68416-R	CGGTTAGGATCCGGTGAGTA	221
18	CV127^[14]	CV127-F	CCTTCGCCGTTTAGTGTATAGG	CV127-R	AGCAGGTTCGTTTAAGGATGAA	238
19	GTS40-3-2^[15]	GTS40-F	TTCAAACCCTTCAATTTAACCGAT	GTS40-R	AAGGATAGTGGGATTGTGCGTC	370
20	A5547^[16]	A5547-F	TGAGGGGGTCAAAGACCAAG	A5547-R	CCAGTCTTTACGGCGAGT	317
21	356043^[17]	356043-F	CTTTTGCCCGAGGTCGTTAG	356043-R	GCCCTTTGGTCTTCTGAGACTG	145
22	FG72	FG72-F	TCGGGCTGCAGGAATTAATGT	FG72-R	TTTGGAGCAATAAACATGTGATAGC	150
23	MON89788^[18]	89788-F	CTGCTCCACTCTTCCTTT	89788-R	AGACTCTGTACCCTGACCT	223
24	305423^[19]	305423-F	CGTCAGGAATAAAGGAAGTACAGTA	305423-R	GCCCTAAAGGATGCGTATAGAGT	235
25	zSSIIb	zSSIIb-F	CGGTGGATGCTAAGGCTGATG	zSSIIb-R	AAAGGGCCAGGTTCATTATCCTC	88
26	SPS	SPS-F	TTGCGCCTGAACGGATAT	SPS-R	GGAGAAGCACTGGACGAGG	277
27	HMG	HMG-F	TCCTTCCGTTTCCTCGCC	HMG-R	TTCCACGCCCTCTCCGCT	206
28	Sad1	Sad1-F	TGGCCTCTAATCATTGTTATGATG	Sad1-R	TTGAGGTGAGTCAGAATGTTGTTC	282
29	MON810^[20]	MON810-F	CAAGTGTGCCCACCACAGC	MON810-R	GCAAGCAAATTCGGAAATGAA	106
30	MON863^[21]	MON863-F	GCACTCAAAGACCTGGCGAATGA	MON863-R	CCATCTTTGGGACCACTGTCG	411
31	Bt11^[22]	Bt11-F	GCGGAACCCCTATTTGTTTA	Bt11-R	CAAGAAATGGTCTCCACCAAA	93
32	MON89034^[23]	89034-F	GCTGCTACTACTATCAAGCCAATA	89034-R	TGCTTTCGCCTATAAATACGAC	207
33	MIR604^[24]	MIR604-F	TCGCGCGCGGTGTCATCTATG	MIR604-R	CGCGACACACCTCGTTAGTTAA	142
34	TT51^[25]	TT51-F	AGCAGAACTTTAACCCCCGAA	TT51-R	AGAGCCTCGTTGGATTTCTTACAT	274
35	KF6-1	KF6-1-F	CGACAAAAGATCAGGATTTGGG	KF6-1-R	GTTTGGAGTTTAAGAGCGA	303
36	MON1445^[26]	MON1445-F	AATGCTGGATTTTCTGCCTGTG	MON1445-R	TCCAAAAGTCATGCATCATTTCTCA	99
37	MON15985^[27]	MON15985-F	ATTTGATGCACTATGTCTTCGTCTA	MON15985-R	CAATGGAATCCGAGGAGGT	175
38	GT73^[28]	GT73-F	AATAACGCTGCGGACATCTA	GT73-R	CAGCAACATTCTCTGTCAACAA	204
39	T45^[29]	T45-F	TCCCATTTATTTACGGTCAC	T45-R	CCATGGGAATTCATTTACAA	233

序号 Serial number	检测靶标 Detection target	引物/探针名称 Primer/Probe name	引物序列Primer sequence（5'-3'）	预期产物大小 Expected product size/bp
1	lectin	lec-QF	TCCACCCCCATCCACATTT	81
		lec-QR	GGCATAGAAGGTGAAGTTGAAGGA
		lec-QP	HEX-AACCGGTAGCGTTGCCAGCTTCG-BHQ1
2	CV127	127-QF	AACAGAAGTTTCCGTTGAGCTTTAAGAC	88
		127-QR	CATTCGTAGCTCGGATCGTGTAC
		127-QP	FAM-TTTGGGGAAGCTGTCCCATGCCC-BHQ1
3	GTS40-3-2^[15]	GTS-QF	TTCATTCAAAATAAGATCATACATACAGGTT	84
		GTS-QR	GGCATTTGTAGGAGCCACCTT
		GTS-QP	FAM-CCTTTTCCATTTGGG-MGBNFQ
4	MON87701^[30]	87701-QF	CGTTTCCCGCCTTCAGTTTAAA	89
		87701-QR	TGGTGATATGAAGATACATGCTTAGCAT
		87701-QP	FAM-TCAGTGTTTGACACACACACTAAGCGTGCC-BHQ1
5	MON87708	87708-QF	TCATACTCATTGCTGATCCATGTAG	91
		87708-QR	AGAACAAATTAACGAAAAGACAGAACG
		87708-QP	FAM-TCCCGGACTTTAGCTCAAAATGCATGTA-BHQ1
6	MON87705	87705-QF	TTCCCGGACATGAAGCCATTTAC	86
		87705-QR	ACAACGGTGCCTTGGCCCAAAG
		87705-QP	FAM-AAGAGACTCAGGGTGTTGTTATCACTGCGG-BHQ1
7	MON87769	87769-QF	CATACTCATTGCTGATCCATGTAGA	87
		87769-QR	GCAAGTTGCTCGTGAAGTT
		87769-QP	JOE-CCCGGACATGAAGCCATTTACAATTGAC-BHQ1
8	MON89788^[18]	89788-QF	TCCCGCTCTAGCGCTTCAAT	139
		89788-QR	TCGAGCAGGACCTGCAGAA
		89788-QP	JOE-CTGAAGGCGGGAAACGACAATCTG-BHQ1
9	305423	305423-QF	CGTGTTCTCTTTTTGGCTAGC	93
		305423-QR	GTGACCAATGAATACATAACACAAACTA
		305423-QP	FAM-TGACACAAATGATTTTCATACAAAAGTCGAGA-BHQ1
10	356043	356043-QF	GTCGAATAGGCTAGGTTTACGAAAAA	99
		356043-QR	TTTGATATTCTTGGAGTAGACGAGAGTGT
		356043-QP	JOE-CTCTAGAGATCCGTCAACATGGTGGAGCAC-BHQ1
11	FG72	FG72-QF	AGATTTGATCGGGCTGCAGG	70
		FG72-QR	GCACGTATTGATGACCGCATTA
		FG72-QP	HEX-AATGTGGTTCATCCGTCTT-MGBNFQ
12	A2704	A2704-QF	GCAAAAAAGCGGTTAGCTCCT	64
		A2704-QR	ATTCAGGCTGCGCAACTGTT
		A2704-QP	HEX-CGGTCCTCCGATCGCCCTTCC-BHQ1
13	A5547^[16]	A5547-QF	GCTATTTGGTGGCATTTTTCCA	75
		A5574-QR	CACTGCGGCCAACTTACTTCT
		A5547-QP	FAM-CCGCAATGTCATACCGTCATCGTTGT-BHQ1
14	DAS68416	68416-QF	GTACATTAAAAACGTCCGCAATGTGT	130
		68416-QR	GTTTAAGAATTAGTTCTTACAGTTTATTGTTAG
		68416-QP	FAM-TTAAGTTGTCTAAGCGTCAATA-MGBNFQ

序号 Serial number	检测靶标 Detection target	引物/探针名称 Primer/Probe name	引物序列Primer sequence（5'-3'）	预期产物大小 Expected product size/bp
1	lectin	lec-QF	TCCACCCCCATCCACATTT	81
		lec-QR	GGCATAGAAGGTGAAGTTGAAGGA
		lec-QP	HEX-AACCGGTAGCGTTGCCAGCTTCG-BHQ1
2	CV127	127-QF	AACAGAAGTTTCCGTTGAGCTTTAAGAC	88
		127-QR	CATTCGTAGCTCGGATCGTGTAC
		127-QP	FAM-TTTGGGGAAGCTGTCCCATGCCC-BHQ1
3	GTS40-3-2^[15]	GTS-QF	TTCATTCAAAATAAGATCATACATACAGGTT	84
		GTS-QR	GGCATTTGTAGGAGCCACCTT
		GTS-QP	FAM-CCTTTTCCATTTGGG-MGBNFQ
4	MON87701^[30]	87701-QF	CGTTTCCCGCCTTCAGTTTAAA	89
		87701-QR	TGGTGATATGAAGATACATGCTTAGCAT
		87701-QP	FAM-TCAGTGTTTGACACACACACTAAGCGTGCC-BHQ1
5	MON87708	87708-QF	TCATACTCATTGCTGATCCATGTAG	91
		87708-QR	AGAACAAATTAACGAAAAGACAGAACG
		87708-QP	FAM-TCCCGGACTTTAGCTCAAAATGCATGTA-BHQ1
6	MON87705	87705-QF	TTCCCGGACATGAAGCCATTTAC	86
		87705-QR	ACAACGGTGCCTTGGCCCAAAG
		87705-QP	FAM-AAGAGACTCAGGGTGTTGTTATCACTGCGG-BHQ1
7	MON87769	87769-QF	CATACTCATTGCTGATCCATGTAGA	87
		87769-QR	GCAAGTTGCTCGTGAAGTT
		87769-QP	JOE-CCCGGACATGAAGCCATTTACAATTGAC-BHQ1
8	MON89788^[18]	89788-QF	TCCCGCTCTAGCGCTTCAAT	139
		89788-QR	TCGAGCAGGACCTGCAGAA
		89788-QP	JOE-CTGAAGGCGGGAAACGACAATCTG-BHQ1
9	305423	305423-QF	CGTGTTCTCTTTTTGGCTAGC	93
		305423-QR	GTGACCAATGAATACATAACACAAACTA
		305423-QP	FAM-TGACACAAATGATTTTCATACAAAAGTCGAGA-BHQ1
10	356043	356043-QF	GTCGAATAGGCTAGGTTTACGAAAAA	99
		356043-QR	TTTGATATTCTTGGAGTAGACGAGAGTGT
		356043-QP	JOE-CTCTAGAGATCCGTCAACATGGTGGAGCAC-BHQ1
11	FG72	FG72-QF	AGATTTGATCGGGCTGCAGG	70
		FG72-QR	GCACGTATTGATGACCGCATTA
		FG72-QP	HEX-AATGTGGTTCATCCGTCTT-MGBNFQ
12	A2704	A2704-QF	GCAAAAAAGCGGTTAGCTCCT	64
		A2704-QR	ATTCAGGCTGCGCAACTGTT
		A2704-QP	HEX-CGGTCCTCCGATCGCCCTTCC-BHQ1
13	A5547^[16]	A5547-QF	GCTATTTGGTGGCATTTTTCCA	75
		A5574-QR	CACTGCGGCCAACTTACTTCT
		A5547-QP	FAM-CCGCAATGTCATACCGTCATCGTTGT-BHQ1
14	DAS68416	68416-QF	GTACATTAAAAACGTCCGCAATGTGT	130
		68416-QR	GTTTAAGAATTAGTTCTTACAGTTTATTGTTAG
		68416-QP	FAM-TTAAGTTGTCTAAGCGTCAATA-MGBNFQ

检测靶标 Detection target	拷贝数浓度Copy number concentration/（copies·μL^-1）	Ct值Ct value				Ct平均值 Mean of Ct value	SD
检测靶标 Detection target	拷贝数浓度Copy number concentration/（copies·μL^-1）	重复1 Repeat 1	重复2 Repeat 2	重复3 Repeat 3	重复4 Repeat 4	Ct平均值 Mean of Ct value	SD
lectin	8×10⁶	16.33	16.24	16.21	16.15	16.23	0.07
	8×10⁵	19.49	19.47	19.44	19.47	19.47	0.02
	8×10⁴	23.00	22.85	22.87	22.80	22.88	0.09
	8×10³	26.12	26.13	26.09	26.02	26.09	0.05
	8×10²	29.10	29.09	29.07	29.13	29.10	0.02
CV127	8×10⁶	16.49	16.42	16.36	16.34	16.40	0.07
	8×10⁵	19.81	19.71	19.77	19.80	19.77	0.04
	8×10⁴	23.32	23.18	23.07	23.05	23.16	0.12
	8×10³	26.54	26.45	26.44	26.40	26.46	0.06
	8×10²	29.75	29.47	29.52	29.71	29.61	0.14
GTS40-3-2	8×10⁶	16.50	16.33	16.41	16.33	16.39	0.08
	8×10⁵	19.69	19.68	19.64	19.63	19.66	0.03
	8×10⁴	23.00	22.94	22.96	22.89	22.95	0.05
	8×10³	26.15	26.15	26.04	26.11	26.11	0.05
	8×10²	29.12	29.29	29.18	29.26	29.21	0.08
MON87701	8×10⁶	16.52	16.55	16.46	16.57	16.53	0.05
	8×10⁵	19.92	19.86	19.85	19.82	19.86	0.04
	8×10⁴	23.29	23.27	23.18	23.22	23.24	0.05
	8×10³	26.55	26.42	26.49	26.42	26.47	0.06
	8×10²	29.79	29.52	29.61	29.70	29.66	0.12
MON87705	8×10⁶	16.42	16.38	16.39	16.35	16.39	0.03
	8×10⁵	19.77	19.71	19.72	19.81	19.75	0.05
	8×10⁴	23.37	23.19	23.25	23.11	23.23	0.11
	8×10³	26.51	26.42	26.44	26.44	26.45	0.04
	8×10²	29.78	29.57	29.56	29.72	29.66	0.11
MON87769	8×10⁶	16.55	16.48	16.49	16.40	16.48	0.06
	8×10⁵	19.84	19.83	19.88	19.79	19.84	0.04
	8×10⁴	23.22	23.25	23.19	23.17	23.21	0.03
	8×10³	26.60	26.52	26.48	26.52	26.53	0.05
	8×10²	29.70	29.75	29.62	29.82	29.72	0.08
MON89788	8×10⁶	16.49	16.42	16.36	16.34	16.40	0.07
	8×10⁵	19.81	19.71	19.77	19.80	19.77	0.04
	8×10⁴	23.32	23.18	23.07	23.05	23.16	0.12
	8×10³	26.54	26.45	26.44	26.40	26.46	0.06
	8×10²	29.75	29.47	29.52	29.71	29.61	0.14
305423	8×10⁶	16.44	16.48	16.39	16.42	16.43	0.04
	8×10⁵	19.73	19.77	19.79	19.81	19.78	0.03
	8×10⁴	23.25	23.28	23.17	23.13	23.21	0.07
	8×10³	26.48	26.44	26.42	26.47	26.45	0.03
	8×10²	29.69	29.54	29.59	29.67	29.62	0.07
356043	8×10⁶	16.45	16.52	16.42	16.44	16.46	0.04
	8×10⁵	19.72	19.78	19.79	19.82	19.78	0.04
	8×10⁴	23.22	23.14	23.17	23.12	23.16	0.04
	8×10³	26.51	26.47	26.48	26.42	26.47	0.04
	8×10²	29.52	29.72	29.60	29.68	29.63	0.09
FG72	8×10⁶	16.47	16.40	16.42	16.36	16.41	0.05
	8×10⁵	19.77	19.68	19.69	19.73	19.72	0.04
	8×10⁴	23.22	23.08	23.11	23.05	23.12	0.07
	8×10³	26.45	26.49	26.41	26.42	26.44	0.04
	8×10²	29.68	29.47	29.49	29.73	29.59	0.13
A2704	8×10⁶	16.38	16.41	16.48	16.42	16.42	0.04
	8×10⁵	19.61	19.65	19.67	19.62	19.64	0.03
	8×10⁴	23.11	23.05	23.02	22.94	23.03	0.07
	8×10³	26.36	26.46	26.52	26.43	26.44	0.07
	8×10²	29.59	29.57	29.52	29.60	29.57	0.04
A5547	8×10⁶	16.44	16.49	16.38	16.35	16.42	0.06
	8×10⁵	19.88	19.77	19.79	19.78	19.81	0.05
	8×10⁴	23.23	23.28	23.19	23.12	23.21	0.07
	8×10³	26.45	26.54	26.48	26.42	26.47	0.05
	8×10²	29.57	29.74	29.52	29.72	29.64	0.11
DAS68416	8×10⁶	16.50	16.44	16.39	16.33	16.42	0.07
	8×10⁵	19.84	19.82	19.87	19.81	19.84	0.03
	8×10⁴	23.42	23.24	23.07	23.01	23.19	0.18
	8×10³	26.49	26.54	26.45	26.48	26.49	0.04
	8×10²	29.55	29.42	29.57	29.68	29.56	0.11