抗逆大豆IND-ØØ41Ø-5转化体特异性定量PCR检测方法的建立及其标准化

doi:10.3864/j.issn.0578-1752.2023.13.002

Abstract

Abstract:

【Objective】Stress-resistant soybean IND-ØØ41Ø-5 has been authorized as a safety certificate for imported processed raw materials in China. This study aims to develop a specific, accurate, and sensitive real-time quantitative PCR (qPCR) assay for the quantification of the stress-resistant soybean IND-ØØ41Ø-5 event, providing a precise measurement technique for regulating its safety in China. 【Method】18 pairs of primers and probes were designed using Beacon designer 8.0 software for the 5′ end flanking sequence of the stress-resistant soybean IND-ØØ41Ø-5 event, in combination with one pair of primer and probe providing by Instituto de Agrobiotecnologia Rosario (INDEAR) S.A. Subsequently, the specific screening of the primers and probes was performed using real-time PCR technology, and one pair of candidate primer and probe was selected. The reaction system parameters, such as primer and probe concentration, were optimized during the establishing the qPCR method. A specificity test was performed using different test samples. The pure stress-resistant soybean IND-ØØ41Ø-5 genomic DNA was serially diluted into standard solution templates, and standard curves were plotted to investigate the linear dynamic range of the qPCR method. Test samples with copy number ratios of 5%, 1% and 0.1% were prepared by mixing IND-ØØ41Ø-5 genomic DNA with non-GM counterpart to evaluate the accuracy of the qPCR method. The limit of detection (LOD) was detected by using test samples with copy number ratios of 0.05% and 0.025%. The limit of quantification (LOQ) was determined after 16 tests on samples with a copy number ratio of 0.1%. Finally, the technical parameters of qPCR assay for the stress-resistant soybean IND-ØØ41Ø-5 event were determined. The specificity, LOD, LOQ and accuracy of the qPCR method were validated by eight qualified testing laboratories. The repeatability and reproducibility of the qPCR were evaluated by Cochran′s test and Grubbs' test, and the measurement uncertainty of the accuracy samples were pre-evaluated by linear least-square method. 【Result】The RBORD-F1/RBORD-R1/RBORD-P1 primer and probe combination was selected as a candidate to establish the qPCR for the stress-resistant soybean IND-ØØ41Ø-5 event, with an amplified fragment of 138 bp. The optimized reaction system had a final concentration of 0.4 μmol·L^-1 for the primer and 0.2 μmol·L^-1 for the probe. Standard curves of IND-ØØ41Ø-5 and Lectin gene assay showed good linearity with the dynamic range from 33 to 83190 copies of genomic DNA. The qPCR can accurately quantify 5%, 1%, and 0.1% content of IND-ØØ41Ø-5 test samples with less than 25% bias and relative standard deviation (RSD). The LOD was determined to be 0.05%, and the LOQ was 0.1%. After validation by eight qualified laboratories, the results indicated that the method was stable, specific and had good repeatability and reproducibility, with the LOD of 0.05% and LOQ of 0.1%. After pre-evaluating the measurement uncertainty, the content of IND-ØØ41Ø-5 in the five test samples was found to be (0.10±0.02)%, (0.53±0.09)%, (1.05±0.18)%, (2.05±0.34)% and (5.18±0.87)%, respectively. These results demonstrate the accuracy and reliability of the qPCR method established in this study for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event components. 【Conclusion】This study successfully developed a specific, accurate, and sensitive qPCR assay for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event using real-time PCR technology. The results show that method is capable of achieving precise measurement and reliable quantification of IND-ØØ41Ø-5 event components.

Key words: transgenic, stress-resistant soybean IND-ØØ41Ø-5, event-specific, real-time quantitative PCR (qPCR)

LI YunJing, XIAO Fang, WU YuHua, LI Jun, GAO HongFei, ZHAI ShanShan, LIANG JinGang, WU Gang. Establishment and Standardization of Event-Specific Real-Time Quantitative PCR Detection Method of Stress-Resistant Soybean IND-ØØ41Ø-5[J].Scientia Agricultura Sinica, 2023, 56(13): 2443-2460.

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References 36

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转基因混合样品 Transgenic mixed samples	成分 Components
大豆Soybean	GTS40-3-2、MON89788、A5547-127、A2704-12、356043、305423、CV127、MON87701、MON87708、MON87769、MON87705、FG72、DAS81419-2
玉米 Maize	Bt11、Bt176、MON810、MON863、GA21、NK603、T25、TC1507、MON89034、MON88017、59122、MIR604、3272、MON87460、DAS40278-9、4114、MON87427、5307
水稻 Rice	TT51-1、KF-6、KMD-1、M12、KF-8、KF-2、G6H1、T1C-19
棉花 Cotton	MON1445、MON531、MON15985、LLCOTTON25、MON88913、GHB614、COT102
油菜 Oilseed rape	MS1、MS8、RF1、RF2、RF3、T45、Oxy-235、Topas19/2、MON88302、73496

编号 No.	引物/探针名称 Primer/probe names	序列 Sequences (5′-3′)	扩增片段 Amplified fragments (bp)	来源 Sources
19	RBORD-F1	GAGAGAGGTTTTTTCTGTTTAAATTCTCACTTTTT	138	罗萨里奥农业生物技术学院公司 Instituto de Agrobiotecnologia Rosario
	RBORD-R1	TCGTTTCCCGCCTTCAGTTTA
	RBORD-P1	FAM-ATCAGTACCCTCAATCATC-MGBNFQ
Lectin	lec-1215F	GCCCTCTACTCCACCCCCA	118	[21]
	lec-1332R	GCCCATCTGCAAGCCTTTTT
	lec-1269P	FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1

引物终浓度 Primer final concentration (μmol·L^-1)	探针终浓度 Probe final concentration (μmol·L^-1)	实时荧光PCR结果Ct值 Ct values of qPCR			Ct平均值 Mean value of Ct
引物终浓度 Primer final concentration (μmol·L^-1)	探针终浓度 Probe final concentration (μmol·L^-1)	平行1 Parallel 1	平行2 Parallel 2	平行3 Parallel 3	Ct平均值 Mean value of Ct
0.9	0.25	33.13	33.06	33.02	33.07
0.2	0.10	33.39	33.46	33.58	33.48
0.4	0.20	32.73	32.63	32.59	32.65
0.6	0.30	32.46	32.54	32.46	32.49
0.8	0.40	32.41	32.36	32.42	32.40

单株 Single plants	浓度 Concentration (copies/μL)		百分比 Percentages (%)
单株 Single plants	Lectin	IND-ØØ41Ø-5	百分比 Percentages (%)
1	730	718	98.36
2	734	723	98.50
3	729	716	98.22
4	740	766	103.47
5	742	721	97.17
6	732	731	99.86
平均值 Means	735	729	99.26

样品 Samples	IND-ØØ41Ø-5测定含量 Contents of IND-ØØ41Ø-5 event (%)			平均值 Means (%)	偏差 Bias (%)	标准偏差 Standard deviation (SD, %)	相对标准偏差 Relative standard deviation (RSD, %)
样品 Samples	重复1 Repeat 1	重复2 Repeat 2	重复3 Repeat 3	平均值 Means (%)	偏差 Bias (%)	标准偏差 Standard deviation (SD, %)	相对标准偏差 Relative standard deviation (RSD, %)
M1	5.02	4.94	5.12	5.03	0.60	0.089	1.77
M2	1.03	1.02	1.08	1.04	4.38	0.034	3.25
M3	0.10	0.11	0.10	0.11	6.61	0.004	3.53

Establishment and Standardization of Event-Specific Real-Time Quantitative PCR Detection Method of Stress-Resistant Soybean IND-ØØ41Ø-5

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测试结果 Test results	IND-ØØ41Ø-5转化体拷贝数 Copies of IND-ØØ41Ø-5 event	Lectin拷贝数 Copies of Lectin gene	百分含量 Percentage level (%)	偏差 Bias (%)
1	69	60350	0.11	14.08
2	69	59600	0.12	16.29
3	64	56560	0.11	13.88
4	64	54500	0.12	16.72
5	63	52490	0.12	19.34
6	61	49660	0.12	23.76
7	60	49400	0.12	21.78
8	60	48750	0.12	22.11
9	59	47640	0.12	24.03
10	59	47590	0.12	23.62
11	53	46210	0.12	15.04
12	51	45870	0.11	11.82
13	53	45100	0.12	17.63
14	47	44170	0.11	6.72
15	41	42960	0.10	4.40
16	37	41190	0.09	10.25
平均值 Means	—	—	0.11	14.51
标准偏差 Standard deviation (SD, %)	—	—	0.01	—
相对标准偏差 Relative standard deviation (RSD, %)	—	—	8.59	—

样品名称 Samples	8家实验室结果 The results of eight laboratories
样品名称 Samples	Lab1	Lab2	Lab3	Lab4	Lab5	Lab6	Lab7	Lab8
阳性对照 Positive control	P	P	P	P	P	P	P	P
阴性对照 Negative control	N	N	N	N	N	N	N	N
1% 抗逆大豆IND-ØØ41Ø-5 1% stress-resistant soybean IND-ØØ41Ø-5	P	P	P	P	P	P	P	P
其他转基因大豆混样 Other transgenic soybean mixed sample	N	N	N	N	N	N	N	N
转基因玉米混样 Transgenic corn mixed sample	N	N	N	N	N	N	N	N
转基因水稻混样 Transgenic rice mixed sample	N	N	N	N	N	N	N	N
转基因棉花混合样 Transgenic cotton mixed sample	N	N	N	N	N	N	N	N
转基因油菜混样 Transgenic rape mixed sample	N	N	N	N	N	N	N	N
非转基因大豆混样 Non-transgenic soybean mixed sample	N	N	N	N	N	N	N	N
0.05%抗逆大豆IND-ØØ41Ø-5 0.05% stress-resistant soybean IND-ØØ41Ø-5	P	P	P	P	P	P	P	P

实验室 Laboratories	Ct值 Ct values
实验室 Laboratories	平行1 Parallel 1	平行2 Parallel 2	平行3 Parallel 3	平行4 Parallel 4	平行5 Parallel 5	平行6 Parallel 6
Lab1	35.22	33.41	33.51	33.54	33.46	33.55
Lab2	35.39	35.6	35.39	35.2	35.08	35.23
Lab3	34.639	34.455	34.663	35.012	34.59	34.854
Lab4	34.68	33.88	34.00	34.64	34.07	34.72
Lab5	35.34	35.24	35.76	35.83	35.35	35.26
Lab6	35.26	35.24	35.53	36.18	35.67	35.64
Lab7	35.68	35.4	35.28	35.55	35.38	35.73
Lab8	35.38	35.76	35.33	35.56	35.19	35.82

实验室 Laboratories	重复 Repeats	Lectin			IND-ØØ41Ø-5
实验室 Laboratories	重复 Repeats	斜率 Slope	PCR效率 PCR Efficiency (%)	决定系数 R²	斜率 Slope	PCR效率 PCR Efficiency (%)	决定系数 R²
Lab1	1	-3.440	95.30	0.999	-3.480	93.80	0.999
	2	-3.428	95.70	1.000	-3.464	94.40	0.997
	3	-3.404	96.70	0.998	-3.463	94.40	1.000
Lab2	1	-3.349	98.90	0.999	-3.418	96.10	0.998
	2	-3.382	97.50	0.999	-3.362	98.40	0.999
	3	-3.378	97.70	1.000	-3.414	96.30	0.999
Lab3	1	-3.391	97.21	0.999	-3.506	93.12	1.000
	2	-3.327	99.77	1.000	-3.476	94.25	1.000
	3	-3.293	101.23	0.999	-3.499	92.96	1.000
Lab4	1	-3.431	95.60	0.995	-3.404	96.70	0.998
	2	-3.325	99.90	0.992	-3.335	99.50	0.994
	3	-3.405	96.60	0.994	-3.356	98.60	0.993
Lab5	1	-3.556	91.10	0.993	-3.565	90.80	0.998
	2	-3.539	91.70	0.998	-3.563	90.90	0.998
	3	-3.527	92.10	0.999	-3.510	92.70	1.000
Lab6	1	-3.315	100.29	0.999	-3.312	100.43	0.998
	2	-3.381	97.58	0.999	-3.381	97.61	1.000
	3	-3.411	96.42	0.996	-3.477	93.90	0.998
Lab7	1	-3.272	102.13	0.999	-3.263	102.50	0.999
	2	-3.228	104.10	0.999	-3.288	101.42	1.000
	3	-3.318	100.18	0.999	-3.275	102.00	1.000
Lab8	1	-3.117	109.30	0.991	-3.297	101.10	0.998
	2	-3.112	109.60	0.983	-3.349	98.90	0.999
	3	-3.189	105.80	0.986	-3.217	104.60	0.998
平均值 Means		-3.355	98.85	0.996	-3.403	96.89	0.998

实验室 Laboratories	重复 Repeats	测试样品 Test samples
实验室 Laboratories	重复 Repeats	5.00% (%)	5.00% (%)	1.00% (%)	0.50% (%)	0.10% (%)
Lab1	1	5.51	2.50	1.12	0.60	0.12
	2	5.44	2.03	1.05	0.55	0.12
	3	5.76	2.32	1.12	0.55	0.13
Lab2	1	5.36	2.05	1.04	0.56	0.11
	2	5.41	2.00	1.06	0.53	0.10
	3	5.48	2.09	1.01	0.54	0.12
Lab3	1	5.31	2.17	1.09	0.49	0.10
	2	5.39	2.22	1.04	0.54	0.09
	3	5.36	2.07	1.08	0.51	0.10
Lab4	1	4.95	2.01	1.00	0.50	0.10
	2	4.99	1.98	1.00	0.53	0.10
	3	4.98	1.94	1.06	0.53	0.11
Lab5	1	5.21	2.05	1.13	0.56	0.10
	2	5.41	2.15	1.04	0.54	0.10
	3	5.79	2.08	1.06	0.55	0.11
Lab6	1	4.65	1.77	0.92*	0.50	0.08
	2	5.62	2.19	1.16	0.62	0.11
	3	5.13	2.13	1.11	0.55	0.09
Lab7	1	5.38	2.01	0.96	0.46	0.10
	2	5.01	1.85	0.91	0.43	0.09
	3	4.75	1.92	0.97	0.48	0.09
Lab8	1	4.41	1.91	0.95	0.52	0.10
	2	4.46	1.88	1.03	0.50	0.11
	3	4.51	1.95	1.05	0.48	0.11

项目 Items	测试样品预期转基因含量 Test sample expected GM level (%)
项目 Items	5.00	2.00	1.00	0.50	0.10
返回数据的实验室数量 Laboratories having returned valid results	8	8	8	8	8
每个实验室样品数量 Samples per laboratory	5	5	5	5	5
离群值的数量 Number of outlies	0	0	1	0	0
剔除的因素 Reason for exclusion	—	—	科克伦检验 Cochran's test	—	—
平均值 Mean value (%)	5.18	2.05	1.04	0.53	0.10
实验室内重复性相对标准偏差 Relative repeatability standard deviation, RSD_r (%)	4.61	6.18	3.66	5.59	7.75
实验室内重复性标准偏差 Repeatability standard deviation, S_r (%)	0.24	0.13	0.04	0.03	0.01
实验室间再现性相对标准偏差 Relative reproducibility standard deviation, RSD_R (%)	8.34	8.61	6.60	8.86	11.58
实验室间再现性标准偏差Reproducibility standard deviation, S_R (%)	0.43	0.18	0.07	0.05	0.01
偏差（绝对值）Bias (absolute value, %)	0.18	0.05	0.04	0.03	0.005
偏差Bias (%)	3.56	2.64	4.46	5.06	4.99

实验室 Laboratories	LOQ样品测量值 Measured values of LOQ samples
实验室 Laboratories	平行1 Parallel 1 (%)	平行2 Parallel 2 (%)	平行3 Parallel 3 (%)	平均值 Means (%)	实验室内标准偏差 In-laboratory standard deviation (SD_r, %)	实验室内重复性相对标准偏差 In-laboratory relative repeatability standard deviation (RSD_r, %)
Lab1	0.12	0.12	0.13	0.12	0.01	5.63
Lab2	0.11	0.10	0.12	0.11	0.01	7.54
Lab3	0.10	0.09	0.10	0.10	0.01	6.42
Lab4	0.10	0.10	0.11	0.10	0.01	6.89
Lab5	0.10	0.10	0.11	0.11	0.00	1.69
Lab6	0.08	0.11	0.09	0.10	0.02	16.82
Lab7	0.10	0.09	0.09	0.10	0.01	5.30
Lab8	0.10	0.11	0.11	0.11	0.01	5.14
平均值 Means				0.10
实验室间标准偏差 Interlaboratory standard deviation (SD_R, %)	0.01
实验室间再现性相对标准偏差 Interlaboratory relative repeatability standard deviation (RSD_R, %)	8.61

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