Comparison of two methods for the quantitative assessment of genetically modified soybeans

CHEN, Chen; ZHANG, Yan; ZHANG, Rui; ZHANG, Yalun; ZHANG, Tao; ZHANG, Zilun; SHI, Guohua; ZHOU, Wei

doi:10.1590/fst.69921

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Food Science and Technology

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Original Article • Food Sci. Technol 42 • 2022 • https://doi.org/10.1590/fst.69921 copy

Comparison of two methods for the quantitative assessment of genetically modified soybeans

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Genetically modified soybean strains are trade monitoring object by China. Currently, the genetically modified soybean MON89788 has been approved in China to be imported as processing raw material. Therefore, there is an urgent need to establish a method to quantitatively assess MON89788. This study used droplet digital PCR technology to quantitatively detect MON89788. The results showed that the genomic DNA concentration and its copy number of genetically modified soybeans showed a certain linear relationship. The formula was y = 2.5967x + 3.1437, where R2 = 0.999. When the same 4.5% genetically modified sample was contained, the results of the droplet digital PCR ratio and linear methods were 5.88% and 3.599%, respectively. Comparing the two results showed that the droplet digital PCR ratio method has a larger error than the linear relationship method. The novel droplet digital PCR linear relationship method established in this study has high sensitivity and specificity, and thus is a good prospect for quantitative research.

Keywords:
digital PCR; quantitative detection; MON89788

Primer/probe name	Working concentration	Sequence (5’→3’)	Probe labeling
MON89788-F	10 μmol/L	TCCCGCTCTAGCGCTTCAAT	FAM-TAMRA
MON89788-R	10 μmol/L	TCGAGCAGGACCTGCAGAA
MON89788-P	10 μmol/L	CTGAAGGCGGGAAACGACAATCTG
Lectin-F	10 μmol/L	CCTCCTCGGGAAAGTTACAA	VIC- TAMRA
Lectin-R	10 μmol/L	GGGCATAGAAGGTGAAGTT
Lectin-P	10 μmol/L	CCCTCGTCTCTTGGTCGCGCCCTCT

Sample name	ng/μL	Copies/μL #1 #2 #3			Average value (copies/μL)	CV (%)
Genetically modified soybean	5	17.6	18.2	16.6	17.46	4.63
	10	28.5	28.4	29.6	28.83	2.31
	20	49.9	51	54.2	51.7	4.32
	40	108	117	112	112.3	4.01
	60	154	162	151	155.67	3.65
	80	204	204	222	210	4.95
	100	280	256	256	264	5.25

name										Mean	Qualitative results
	Repeat 1			Repeat 2			Repeat 3
	1	2	3	1	2	3	1	2	3
MON89788 foreign gene Ct value	31.23	31.08	31.42	31.55	31.45	31.26	31.77	32.01	32.14	31.55	Transgene positive
Ct value of soybean endogenous gene	27.40	27.39	27.37	27.51	27.52	27.52	28.52	28.39	28.36	27.78	Transgene positive

name	Repeat experiment
	Repeat 1			Repeat 2			Repeat 3			Mean	RSD
	1	2	3	1	2	3	1	2	3	Mean	RSD
MON89788 Foreign gene copy number	12.9	12.5	14.6	12.5	14.6	10.7	12.1	11	11.5	12.49	11.19%
Soybean endogenous gene copy number	240	232	238	208	207	194	204	194	195	212.44	8.96%
Exogenous gene/endogenous gene	5.38%	5.39%	6.13%	6.01%	7.05%	5.52%	5.93%	5.67%	5.90%	5.88%	8.79%

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[1] *Corresponding author: shiguohua@nepp.com.cn; zhouwei0311@163.com