羊源性基因组DNA标准物质研制

doi:10.19586/j.2095-2341.2023.0137

生物技术进展 ›› 2024, Vol. 14 ›› Issue (1): 125-132.DOI: 10.19586/j.2095-2341.2023.0137

• 研究论文 • 上一篇

羊源性基因组DNA标准物质研制

纪艺¹(), 王凯莉², 余卉茹³, 赵新⁴, 丁霖¹, 彭城¹, 徐俊锋¹, 陈笑芸¹()

^1.浙江省农业科学院，农产品质量安全危害因子与风险防控国家重点实验室，农业农村部农业转基因生物溯源重点实验室，杭州 310021
^2.宁波大学食品与药学学院，浙江宁波 215211
^3.湘湖实验室，杭州 311231
^4.天津市农业科学院，种质资源与生物技术研究所，天津 300384

收稿日期:2023-10-25 接受日期:2023-11-25 出版日期:2024-01-25 发布日期:2024-02-05
通讯作者: 陈笑芸
作者简介:纪艺E-mail: jymemory12138@163.com;
基金资助:
科技创新专项2030重大项目农业生物育种重大专项(2022ZD0402012);浙江省重点研发项目(2021C02059)

Development of Sheep-derived Genomic DNA Reference Material

Yi JI¹(), Kaili WANG², Huiru YU³, Xin ZHAO⁴, Lin DING¹, Cheng PENG¹, Junfeng XU¹, Xiaoyun CHEN¹()

^1.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products，Key Laboratory of Traceability for Agricultural Genetically Modified Organisms，Ministry of Agriculture and Rural Affairs，Zhejiang Academy of Agricultural Sciences，Hangzhou 310021，China
^2.School of Food and Pharmacy，Ningbo University，Zhejiang Ningbo 215211，China
^3.Xianghu Laboratory，Hangzhou 311231，China
^4.Institute of Germplasm Resources and Biotechnology，Tianjin Academy of Agricultural Sciences，Tianjin 300384，China

Received:2023-10-25 Accepted:2023-11-25 Online:2024-01-25 Published:2024-02-05
Contact: Xiaoyun CHEN

摘要/Abstract

摘要：

近年来，羊肉及其肉制品的掺假等食品安全事件层出不穷，为了完善市场执法检查法律依据，利用数字PCR定值羊HELZ基因，研制了羊基因组DNA标准物质。由于标准物质可以用于衡量检测方法的准确性，因此可以判定食品及相关制品中羊肉的掺假情况。利用数字PCR对研制的标准物质进行均匀性和稳定性评估，结果表明该批标准物质均匀性良好，在4 ℃、25 ℃可以稳定保存14 d，在-20 ℃可以稳定保存6个月。来自全国9个不同实验室羊源性基因组DNA标准物质（高浓度）和羊源性基因组DNA标准物质（低浓度）的联合定值结果显示，标准值及其扩展不确定度分别为（5.44±0.45）×10³ copies·μL^-1和（5.68±0.54）×10² copies·μL^-1。该羊源性基因组DNA标准物质为动物源性标准物质的市场应用提供了技术基础，完善了羊源性基因组DNA标准物质的制备、定量检测、质量控制和量值溯源技术平台。

关键词: 标准物质, 羊源性HELZ基因, 数字PCR

Abstract:

In recent years， food safety incidents such as adulteration of mutton and its meat products have been occurring， and in order to improve the legal basis for market enforcement and inspection， a standard substance for sheep genomic DNA has been developed by using digital PCR to quantify the sheep HELZ gene. Since the standard substance can be used to measure the accuracy of detection methods， it can determine the adulteration of mutton in food and related products. In this research， the standard material of sheep genome DNA was developed as the positive control for the detection of mutton in food and related products. The uniformity and stability of the reference materials were evaluated by digital PCR. The results showed that the reference materials had good uniformity and could be stably stored at 4 ℃ and 25 ℃ for 14 days， and at -20 ℃ for 6 months. The standard values of sheep-derived genomic DNA reference material （high concentration） and sheep-derived DNA reference material （low concentration） and their extended uncertainties were （5.44±0.45） ×10³ copies·μL^-1 and （5.68±0.54） ×10² copies·μL^-1， respectively， which were jointly determined by nine different laboratories. The development of sheep-derived genomic DNA reference materials provides a technical basis for the development of animal-derived reference materials， and will establish a technical platform for the preparation， quantitative detection， quality control and quantitative value traceability of sheep-derived genomic DNA reference materials.

Key words: reference material, HELZ gene of sheep-derived, digital PCR

中图分类号:

Q789

纪艺, 王凯莉, 余卉茹, 赵新, 丁霖, 彭城, 徐俊锋, 陈笑芸. 羊源性基因组DNA标准物质研制[J]. 生物技术进展, 2024, 14(1): 125-132.

Yi JI, Kaili WANG, Huiru YU, Xin ZHAO, Lin DING, Cheng PENG, Junfeng XU, Xiaoyun CHEN. Development of Sheep-derived Genomic DNA Reference Material[J]. Current Biotechnology, 2024, 14(1): 125-132.

图/表 10

图1 羊源性DNA电泳检测图注：M—DNA marker。

Fig. 1 Electrophoretic detection of sheep DNA

表1 均匀性初检统计分析结果

Table 1 Results of statistical analysis of the uniformity

标准物质	组间自由度 $v 1$	组内自由度 $v 2$	组间均方 $M 组间$	组内均方 $M 组内$	统计量(F)	F_0.05(5,12)
HELZ（高浓度）	5	12	1.48×10⁴	9.90×10³	1.49	3.11
HELZ（低浓度）	5	12	1.41×10²	1.29×10²	1.09	3.11

表1 均匀性初检统计分析结果

Table 1 Results of statistical analysis of the uniformity

标准物质	组间自由度 $v 1$	组内自由度 $v 2$	组间均方 $M 组间$	组内均方 $M 组内$	统计量(F)	F_0.05(5,12)
HELZ（高浓度）	5	12	1.48×10⁴	9.90×10³	1.49	3.11
HELZ（低浓度）	5	12	1.41×10²	1.29×10²	1.09	3.11

表2 均匀性统计分析结果

Table 2 Uniformity statistical analysis results

标准物质	组间自由度 $v 1$	组内自由度 $v 2$	组间均方 $M 组间$	组内均方 $M 组内$	$u b b = s b b$	$u b b, r e l$	F_0.05(11,24)
HELZ（高浓度）	11	24	1.05×10⁴	1.03×10²	54.23	1.1%	2.215
HELZ（低浓度）	11	24	2.96×10²	2.06×10¹	7.96	1.5%	2.215

表2 均匀性统计分析结果

Table 2 Uniformity statistical analysis results

标准物质	组间自由度 $v 1$	组内自由度 $v 2$	组间均方 $M 组间$	组内均方 $M 组内$	$u b b = s b b$	$u b b, r e l$	F_0.05(11,24)
HELZ（高浓度）	11	24	1.05×10⁴	1.03×10²	54.23	1.1%	2.215
HELZ（低浓度）	11	24	2.96×10²	2.06×10¹	7.96	1.5%	2.215

表3 短期稳定性评估结果

Table 3 Short-term stability assessment results

标准物质	HELZ（高浓度）		HELZ（低浓度）
标准物质	4 ℃	25 ℃	4 ℃	25 ℃
总均值	5.28×10³	5.25×10³	5.68×10²	5.66×10²
β₁	3.70	-4.82	-0.48	-0.27
截距β₀	5 258.37	5 281.60	571.26	567.36
标准偏差S(β₁)	4.42	4.48	0.76	0.81
t_0.95,2	4.3	4.3	4.3	4.3
t_0.95,2×S(β₁)	19.006	19.264	3.268	3.483
稳定性判断	│β₁│<t_0.95,3×S(β₁)
判断结果	稳定	稳定	稳定	稳定

图2 标准物质在4、25 ℃短期稳定性趋势图

Fig. 2 Short-term stability trend of reference material at 4 and 25 ℃

表4　-20 ℃长期稳定性评估结果

Table 4 Evaluation results of long-term stability at -20 ℃

标准物质	HELZ（高浓度）	HELZ（低浓度）
均值	5.21×10³	5.43×10²
β₁	7.92	0.32
截距β₀	5 192.56	562.72
标准偏差S(β₁)	15.07	1.61
t_0.95,3	3.18	3.18
t_0.95,3×S(β₁)	47.90	5.12
稳定性判断	│β₁│<t_0.95,3×S(β₁)
判断结果	稳定	稳定

图3 标准物质-20 ℃长期稳定性趋势图

Fig. 3 Long-term stability trend of reference material at -20 ℃

表5 冻融稳定性评估结果

Table 5 Evaluation results of freeze-thaw stability

标准物质	HELZ（高浓度）	HELZ（低浓度）
均值	5.24×10³	5.69×10²
β₁	9.37	-0.40
截距β₀	5 183.78	570.80
标准偏差S(β₁)	5.06	0.93
t_0.95,8	2.31	2.31
t_β1	1.85	0.43
稳定性判断	t_β1<t_0.95,8
判断结果	稳定	稳定

表6 9家实验室联合定值数据分析

Table 6 Analysis of joint setting data from 9 laboratories

实验室编号	Copies·μL^-1
实验室编号	HELZ(高浓度）	HELZ(低浓度）
1	5.18×10³	5.40×10²
2	5.59×10³	5.83×10²
3	5.71×10³	5.87×10²
4	5.45×10³	5.55×10²
5	5.52×10³	5.78×10²
6	5.33×10³	5.69×10²
7	5.41×10³	5.56×10²
8	5.35×10³	5.74×10²
9	5.41×10³	5.68×10²
总平均值	5.44×10³	5.68×10²
SD	1.55×10²	1.51×10¹
RSD/%	2.85	2.66

表7 不确定度评定结果

Table 7 Uncertainty evaluation results

标准物质	标准值Y	定值相对不确定度 $u r e l, c$	均匀性相对不确定度 $u r e l, b b$	短期稳定性相对不确定度 $u r e l, s s$	长期稳定性相对不确定度 $u r e l, l s$	相对标准不确定度 $u r e l, C R M$	相对扩展不确定度 $u r e l, C R M$	扩展不确定度U_CRM (k=2)/(copies·μL^-1)
HELZ(高浓度)	5.44×10³	0.033	0.011	0.012	0.018	0.041	0.082	0.45×10³
HELZ(低浓度)	5.68×10²	0.035	0.015	0.020	0.018	0.047	0.094	0.54×10²

表7 不确定度评定结果

Table 7 Uncertainty evaluation results

标准物质	标准值Y	定值相对不确定度 $u r e l, c$	均匀性相对不确定度 $u r e l, b b$	短期稳定性相对不确定度 $u r e l, s s$	长期稳定性相对不确定度 $u r e l, l s$	相对标准不确定度 $u r e l, C R M$	相对扩展不确定度 $u r e l, C R M$	扩展不确定度U_CRM (k=2)/(copies·μL^-1)
HELZ(高浓度)	5.44×10³	0.033	0.011	0.012	0.018	0.041	0.082	0.45×10³
HELZ(低浓度)	5.68×10²	0.035	0.015	0.020	0.018	0.047	0.094	0.54×10²

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羊源性基因组DNA标准物质研制

Development of Sheep-derived Genomic DNA Reference Material

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