Development of plasmid DNA reference material of genetically modified maize TC1507

doi:10.3969/j.issn.1004-1524.2021.03.02

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (3): 390-395.DOI: 10.3969/j.issn.1004-1524.2021.03.02

• Crop Science • Previous Articles Next Articles

Development of plasmid DNA reference material of genetically modified maize TC1507

QU Zhan, YANG Litao^*()

School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2020-06-04 Online:2021-04-02 Published:2021-03-25
Contact: YANG Litao

Abstract

Abstract:

The reference material has three typical characteristics of characterization value, homogeneity and stability, which was used as the scale of biological measurement. The development of reference materials for genetically modified organisms was one of the key technologies for the implementation of the GMO labeling system in China. In this study, a novel plasmid DNA standard substance pTC1507 specific to the transgene maize TC1507 was produced, and its homogeneity, stability and characterization were evaluated and measured. The results showed that the pTC1507 had good homogeneity between and within bottles. The stability of pTC1507 was reliable, and it could be stably placed at -20 ℃ for 6 months at least. After sequencing, quantitative real-time PCR analysis, and uncertainty evaluation, the characterization value of pTC1507 was 1.01±0.053. The results of homogeneity, stability and characterization evaluation showed that pTC1507 was satisfied with the typical requirements of reference materials and could replace the conventional matrix-based reference materials in the detection of transgenic maize. The developed plasmid reference material showed the advantages of easy producing, simple preparation, and low cost.

Key words: genetically modified maize, TC1507, plasmid DNA reference material, characterization value

CLC Number:

S513

QU Zhan, YANG Litao. Development of plasmid DNA reference material of genetically modified maize TC1507[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 390-395.

Figures/Tables 8

Table 1 Primers and probes used in genetically modified maize TC1507 plasmid reference material production

靶标基因	名称	序列	扩增子
Target gene	Primer name	Sequence(5'-3')	Amplicon/bp
TC1507	Q-TC1507-1F	GACGTCTCAATGTAATGGTTAACGA	83
	Q-TC1507-2R	CCTAGTATATGAAAGAATGAAAAGGTGCTT
	Q-TC1507-P	FAM-TCACAAACCGCGGAACACAAGAACG-TAMRA
zSSIIb	Q-zSSIIb-1F	CGGTGGATGCTAAGGCTGATG	88
	Q-zSSIIb-2R	AAAGGGCCAGGTTCATTATCCTC
	Q-zSSIIb-P	FAM-TAAGGAGCACTCGCCGCCGCATCTG-BHQ1

Fig.1 The schematic diagram of pTC1507

Fig.2 Enzyme digestion of pTC1507 plasmid Lane 1, 3 represented double enzyme digestion electrophoretogram with EcoRⅠ/XbaⅠ; Lane 2, 4 represented double enzyme digestion electrophoretogram with NcoRⅠ/SpeⅠ; M, DL2000 DNA marker.

Table 2 Variance analysis of plasmid reference material TC1507 homogeneity

TC1507		zSSIIb
总的Ct平均值	18.38	总的Ct平均值	19.85
Total Ct value		Total Ct value
平均拷贝数	5 138 401	平均拷贝数	5 163 483
Mean copy number		Mean copy number
总的标准偏差	0.05	总的标准偏差	0.05
Total standard deviation		Total standard deviation
总的相对标准偏差	0.25	总的相对标准偏差	0.24
Total relative standard deviation/%		Total relative standard deviation/%
组间平方和	0.091 2	组间平方和	0.093 9
Quadratic sum between groups		Quadratic sum between groups
γ1	14	γ1	14
组内平方和	0.204 3	组内平方和	0.185 7
Quadratic sum within group		Quadratic sum within group
γ2	30	γ2	30
F	0.96	F	1.08
F_α	2.01	F_α	2.01
比较结果	F<F_α	比较结果	F<F_α
Comparative		Comparative
结论	样品均匀性良好
Conclusion	The sample was good in homogeneity

Fig.3 Short term stability evaluation of plasmid reference material pTC1507

Fig.4 Long term stability evaluation of plasmid reference material pTC1507

Table 3 Certified value of plasmid reference material TC1507 based on copy number

理论拷贝数 Theoretical copy number	TC1507转化体特异性序列实际拷贝数 True copy number of TC1507 specific sequence				玉米内源zSSIIb序列实际拷贝数 True copy number of zSSIIb sequence				拷贝数比值 Copy number ratio			平均值± 标准偏差 Mean± Standard deviation
理论拷贝数 Theoretical copy number	1	2	3	平均拷贝数 Mean copy number	1	2	3	平均拷贝数 Mean copy number	1	2	3	平均值± 标准偏差 Mean± Standard deviation
500000	550 736	508 271	5×10⁵	517 554	544 427	548 352	478 387	523 722	1.01	0.9	1.03	1.01±0.053
50000	54 140	44 138	44 138	47 474	49 415	49 771	45 660	48 282	1.09	0.9	0.97
5000	4 911	5 020	4 500	4 810	4 582	4 784	4 716	4 694	1.07	1	0.95
500	468	442	465	458	453	443	440	445	1.03	1	1.05
50	64	62	63	64	61	63	60	61	1.05	1	1.05

Table 4 Certified value and uncertainty of pTC1507 plasmid reference material

不确定度来源 Uncertainty source	m	n	$S 12$	$S 22$	$S char / H / T 2$
u_char	6	9			0.000 05
S_H (TC1507)	15	9	0.019 38	0.002 45	0.001 88
S_H (zSSIIb)	15	9	0.020 11	0.002 45	0.001 96
S_T (TC1507)	4	9	0.004 22	0.000 42	0.000 04
S_T (zSSIIb)	4	9	0.008 04	0.001 46	0.000 07
u_CRM	0.06

Table 4 Certified value and uncertainty of pTC1507 plasmid reference material

不确定度来源 Uncertainty source	m	n	$S 12$	$S 22$	$S char / H / T 2$
u_char	6	9			0.000 05
S_H (TC1507)	15	9	0.019 38	0.002 45	0.001 88
S_H (zSSIIb)	15	9	0.020 11	0.002 45	0.001 96
S_T (TC1507)	4	9	0.004 22	0.000 42	0.000 04
S_T (zSSIIb)	4	9	0.008 04	0.001 46	0.000 07
u_CRM	0.06

References 10

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Development of plasmid DNA reference material of genetically modified maize TC1507

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