转基因玉米MON87427梯度含量基体标准物质的研制

doi:10.3864/j.issn.0578-1752.2023.08.002

摘要/Abstract

摘要：

【目的】转基因检测标准物质是转基因生物安全监管、标识制度实施的物质基础，是实验室内检测数据溯源、实验室间检测数据可比的保证。中国于2017年批准进口转基因玉米MON87427用作加工原料，其安全监管和检测急需研制有证标准物质。【方法】以开发商提供的转基因玉米MON87427杂合种子和非转基因受体种子为原材料，对转基因玉米MON87427和其受体种子分别清洗、干燥、冷冻研磨、粒径及含水量检测，按质量分数（以干基计）称量配制得到质量分数分别为60.0、99.5和1 000.0 mg·g^-1的转基因玉米MON87427基体标准物质MON87427a、MON87427b和MON87427c。运用实时荧光定量PCR进行粉末的均匀性初检，初检合格后分装。用MON87427/zSSIIb二重微滴数字PCR（ddPCR）方法进行标准物质均匀性、稳定性评估和8家实验室联合定值。根据《标准物质定值的通用原则及统计学原理》（JJF 1343）进行标准物质均匀性评估、稳定性评估、联合定值数据的处理及不确定度评定。【结果】本批标准物质有MON87427a、MON87427b、MON87427c 3个浓度，粒径小于200 μm的粉末大于80%，含水量低于5%。用棕色玻璃瓶分装，不低于1.0 g/瓶，充氮后轧盖，3个浓度的标准物质各分装400瓶。3种标准物质均匀性检验的F值均小于临界值F_0.05(14,30)（2.04），在瓶内和瓶间具有良好的均匀性，使用时，最小取样量为100 mg。标准物质在25℃、37℃和60℃条件下可稳定储存14 d，表明在常温下运输14 d标准物质特性量值不发生趋势性变化；在4℃和-20℃条件下长期稳定性达到12个月；开盖取样5次，标准物质特性量值未发生显著偏离。8家实验室联合测定的标准物质拷贝数比值无异常值、呈正态分布、且等精度，标准物质MON87427a、MON87427b、MON87427c的标准值及扩展不确定度分别为（2.92±0.44）%、（4.89±0.57）%、（52.1±3.4）%。【结论】研制的转基因玉米MON87427梯度含量基体标准物质均匀性良好，可稳定运输和储存，满足转基因玉米MON87427定性、定量检测需求，为转基因玉米MON87427的安全监管、定量标识制度的实施提供了可靠的有证标准物质。

关键词: 转基因玉米MON87427, 基体标准物质, 二重微滴数字PCR, 特性量值, 不确定度

Abstract:

【Objective】The GMO (genetically modified organism) reference materials (RMs) are the material basis for GMO safety supervision and labeling policy implementation. During GMO detection the utilization of RMs guarantees the traceability of quantitative results within laboratory and the comparability of quantitative results between laboratories. GM (genetically modified) maize MON87427 has been approved to be imported as raw material in China, it’s urgent to develop certified reference materials (CRMs) for safety supervision and quantification. 【Method】 The GM maize MON87427 hybrid seeds and non-GM counterparts provided by the developer were used as raw materials to perform washing, drying, freeze-grinding, particle size measurement, and moisture content measurement, sequentially. The matrix RMs of MON87427a, MON87427b and MON87427c were produced by blending the seed powder of the GM maize MON87427 and a non-GM counterpart in GMO mass fractions of 60.0 mg·g^-1, 99.5 mg·g^-1 and 1 000.0 mg·g^-1 on a dry basis. The real-time quantitative PCR was used to conduct an initial assessment of homogeneity before packing. The MON87427/zSSIIb duplex digital PCR (ddPCR) was used to evaluate the homogeneity and stability of the RMs as well as the collaborative characterization by 8 qualified laboratories. The data processing of homogeneity, stability, collaborative characterization together with uncertainty evaluation of RMs were carried out according to the standard "General and Statistical Principles for Characterization of Reference Materials" (JJF 1343). 【Result】 This batch of RMs contains three RMs of MON87427a, MON87427b, and MON87427c in different mass fractions, with more than 80% of particle size of less than 200 μm and less than 5% of moisture content. The RMs were packed in brown glass bottles, nitrogen flushed before capping, bottling amount was not less than 1.0 g/bottle, a total of 400 bottles were produced for each mass fraction RM. The calculated F values of the homogeneity test were all less than the critical value of F_{0.05 (14, 30)} (2.04) for the three RMs, displaying good homogeneity within and between bottles, and the minimum intake was determined to be 100 mg. The RMs can be stored stably at 25℃, 37℃, and 60℃ for 14 days, the property value of the RMs does not change significantly after 14 days of transportation at room temperature; the long-term stability can reach 12 months at 4℃ and -20℃; The property value of the samples taken from the same bottle of RM after 5 opening-capping cycles, does not deviate significantly from that of the first taken sample. The collaborative characterization data by eight qualified laboratories displayed normal distribution without outliers and outlying standard deviations. The standard value and expanded uncertainty of MON87427a, MON87427b, Mon87427c were certified to be (2.92±0.44)%, (4.89±0.57)%, (52.1±3.4)%. 【Conclusion】The developed GM maize MON87427 matrix RMs in different mass fractions have good homogeneity, and can be stably transported and stored. This batch of RMs meets the requirements of qualitative and quantitative detection of MON87427 event, providing reliable CRMs for the safety supervision and implementation of quantitative labeling policy for GMO-derived products.

Key words: genetically modified maize MON87427, matrix reference material, duplex digital PCR, property value, uncertainty

李俊, 单露英, 肖芳, 李允静, 高鸿飞, 翟杉杉, 吴刚, 张秀杰, 武玉花. 转基因玉米MON87427梯度含量基体标准物质的研制[J]. 中国农业科学, 2023, 56(8): 1444-1455.

LI Jun, SHAN LuYing, XIAO Fang, LI YunJing, GAO HongFei, ZHAI ShanShan, WU Gang, ZHANG XiuJie, WU YuHua. Development of A Set of Matrix Reference Materials in Different Mass Fractions of Genetically Modified Maize MON87427[J]. Scientia Agricultura Sinica, 2023, 56(8): 1444-1455.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.08.002

https://www.chinaagrisci.com/CN/Y2023/V56/I8/1444

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PCR类型 PCR type	靶标 Target	引物/探针序列 Sequence of primer/probe (5′-3′)	扩增片段大小 Amplicon size (bp)
实时荧光定量PCR Real-time PCR	MON87427	F: ACGGAAACGGTCGGGTCAAATG	95
		R: CCATGTAGATTTCCCGGTTTTCTC
		P: FAM- TCGGGACAATATGGAGAAAAAGAAAGAG -BHQ1
	zSSIIb	F: CGGTGGATGCTAAGGCTGATG	88
		R: AAAGGGCCAGGTTCATTATCCTC
		P: Hex-TAAGGAGCACTCGCCGCCGCATCTG-BHQ1
普通PCR Conventional PCR	MON87427	F: ACGGAAACGGTCGGGTCAAATG	95
	MON87427	R: CCATGTAGATTTCCCGGTTTTCTC	95
	插入位点 Insert site	87427CH-F: ATAAGTCCTCTCCCGTTTCCGT	416
	插入位点 Insert site	87427CH-R: TCGATGGGTCGAGAAGATTC	416

标准物质 Reference materials	拟称取质量 Proposed weighing mass (g)			实际称取质量 Practical weighing mass (g)			含水量 Water content (g)		转基因含量 GMO content (mg·g^-1)
标准物质 Reference materials	总重量 Total mass	转基因粉末 GM powder	非转基因粉末 Non-GM powder	总重量 Total mass	转基因粉末 GM powder	非转基因粉末 Non-GM powder	转基因粉末 GM powder	非转基因粉末 Non-GM powder	转基因含量 GMO content (mg·g^-1)
MON87427a	450	26.995	423.005	450.002	27.005	422.997	0.292	4.653	60.02
MON87427b	450	44.992	405.008	450.000	44.778	405.222	0.484	4.457	99.52

标准物质 Reference materials	MON87427a			MON87427b			MON87427c
标准物质 Reference materials	12 h	18 h	24 h	12 h	18 h	24 h	12 h	18 h	24 h
平均值 Mean	3.31	3.25	3.33	5.44	5.23	5.24	51.22	51.84	52.20
标准差 SD	0.21	0.12	0.08	0.63	0.35	0.17	1.77	1.34	2.09
相对标准差 Relative SD	6.40	3.54	2.51	11.55	6.68	3.26	3.45	2.59	4.01
统计量F Statistic F	1.60	1.73	2.18	2.17	0.56	0.74	1.31	2.32	1.89
临界值 F_0.05(8,18)	2.51	2.51	2.51	2.51	2.51	2.51	2.51	2.51	2.51
结论 Conclusion	均匀 Homogeneous			均匀 Homogeneous			均匀 Homogeneous

标准物质 <BOLD>R</BOLD>eference materials	差来源 Variance source	差方和（Q） Sum of squares of deviation	自由度（f） Degree of freedom	方差（S） Variance	统计量（F） Statistic F	F_{0.05 (14,30)}	比较结果 Comparison	结论 Conclusion
MON87427a	瓶间 Between-vial	0.25	14	0.018	1.70	2.04	F<F_{0.05 (14,30)}	均匀 Homogeneous
MON87427a	瓶内 Within-vial	0.31	30	0.010	1.70		F<F_{0.05 (14,30)}	均匀 Homogeneous
MON87427b	瓶间 Between-vial	0.56	14	0.040	1.40		F<F_{0.05 (14,30)}	均匀 Homogeneous
MON87427b	瓶内 Within-vial	0.85	30	0.028	1.40		F<F_{0.05 (14,30)}	均匀 Homogeneous
MON87427c	瓶间 Between-vial	11.97	14	0.855	1.63		F<F_{0.05 (14,30)}	均匀 Homogeneous
MON87427c	瓶内 Within-vial	15.75	30	0.525	1.63		F<F_{0.05 (14,30)}	均匀 Homogeneous

标准物质 <BOLD>R</BOLD>eference materials	MON87427a			MON87427b			MON87427c
标准物质 <BOLD>R</BOLD>eference materials	25℃	37℃	60℃	25℃	37℃	60℃	25℃	37℃	60℃
平均值Mean	2.77	2.76	2.87	4.80	4.63	4.63	49.73	50.53	50.14
斜率(β₁)Slope	-0.0122	0.0085	0.0031	-0.0095	-0.0127	-0.0045	-0.0666	0.0650	-0.0443
与斜率相关的不确定度s(β₁)	0.0092	0.0026	0.0129	0.0062	0.0051	0.0069	0.0284	0.0656	0.0882
t_0.95,_n-2	4.3027	4.3027	4.3027	4.3027	4.3027	4.3027	4.3027	4.3027	4.3027
t_0.95,_n-2* s(β₁)	0.0397	0.0111	0.0556	0.0268	0.0218	0.0297	0.1224	0.2823	0.3793
稳定性判断 Comparison	\|β₁\| <t_0.95_n_-2·s(β₁)
判断结果 Conclusion	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable	稳定 Stable