Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (22): 4728-4739.doi: 10.3864/j.issn.0578-1752.2021.22.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment and Application of A Duplex ddPCR Method to Quantify the NK603/zSSIIb Copy Number Ratio in Transgenic Maize NK603

XIAO Fang1(),LI Jun1,WANG HaoQian2,ZHAI ShanShan1,CHEN ZiYan2,GAO HongFei1,LI YunJing1,WU Gang1,ZHANG XiuJie2(),WU YuHua1()   

  1. 1Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement Oil Crops Ministry of Agriculture and Rural Affairs, Wuhan 430062
    2Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs of People’s Republic of China, Beijing 100025
  • Received:2021-04-28 Accepted:2021-06-16 Online:2021-11-16 Published:2021-11-19
  • Contact: XiuJie ZHANG,YuHua WU E-mail:xf_hzau@163.com;zhxj7410@sina.com;wuyuhua@oilcrops.cn

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Abstract:

【Objective】Transgenic maize NK603 approved for import is an important target of genetically modified organism (GMO) regulation in China. The implementation of GMO regulation requires reference materials (RMs) and standardized detection methods. Establishment of a duplex droplet digital PCR (ddPCR) would provide accurate measurement technology for quantification of NK603 event and development of NK603 RMs. 【Method】 A standard plasmid molecule pUC57-NK603 was constructed by DNA synthetic technique; the primer/probe set of NK603 event was combined with different maize reference genes one by one to select reference gene PCR assay with identical amplification ability to NK603 event-specific PCR assay; main reaction parameters, such as annealing temperature and primer/probe concentration,were optimized in the course of establishing duplex ddPCR; the standard plasmid solution was serially diluted to investigate the limit of detection (LOD), limit of quantification (LOQ) and dynamic range of the duplex ddPCR assay; blinded samples with mass fractions of 100%, 10% and 6% were prepared by mixing NK603 powder with non-GM counterpart, to evaluate the accuracy of quantitative results of the duplex ddPCR. 【Result】 The reference gene zSSIIb was determined to combine with NK603 event to establish the NK603/zSSIIb duplex ddPCR method with the standard plasmid molecule pUC57-NK603 as a quality control after analyzing the fluorescence amplitude of positive droplets, separation between positive and negative droplets, raindrop number, and consistency between measured copy number ratio and expected copy number ratio of NK603 event to reference gene. The primer/probe concentration was optimized to be 400 nmol·L-1/200 nmol·L-1 for both NK603 event and zSSIIb gene, and the annealing temperature was determined to be 60°C. The LOD of NK603/zSSIIb duplex ddPCR was estimated to be 2 copies of DNA template, the LOQ was 48 copies of DNA template, both NK603 assay and zSSIIb assay showed good linearity between measured values and theoretical values over the dynamic range from 10 to 60 000 copies of DNA template. The NK603/zSSIIb duplex ddPCR achieved accurate quantitative results of NK603 content in blind maize samples with less than 25% of coefficient of variation; the quantitative results of ddPCR were not significantly different from those of real-time quantitative PCR (qPCR), moreover, the duplex ddPCR showed an advantage over qPCR in term of precision. 【Conclusion】 The selection of reference genes affects the accuracy of quantitative results by ddPCR. Establishment of ddPCR methods should use samples with accurate GMO content as quality controls to evaluate the applicability of reference genes. The NK603/zSSIIb duplex ddPCR method was successfully established using the synthetic standard plasmid molecule pUC57-NK603 as a quality control. NK603 certified reference materials (CRMs) have been successfully developed in China by applying the established NK603/zSSIIb duplex ddPCR assay to characterize the property values.

Key words: transgenic maize NK603, duplex droplet digital PCR, reference plasmid molecule, reference gene, quantification

Table 1

Primer and probe information for PCR assays"

靶标
Target		 引物/探针名称
Primer/probe name		 序列
Sequence (5'-3')		 片段长度
Fragment size (bp)		 参考文献
Reference
NK603转化体
NK603 event		 603-QF ATGAATGACCTCGAGTAAGCTTGTTAA 108 [30]
603-QR AAGAGATAACAGGATCCACTCAAACACT
603-QP TGGTACCACGCGACACACTTCCACTC
zein-ENDO zein-ENDO F GCATTGTTCGCTCTCCTAGC 110 [31]
zein-ENDO R TACTGCATCCATGGGTTCAT
zein-ENDO P TTCCAGGGCACTTGCCACCA
zein-taq zein-taqze F GCCATTGGGTACCATGAACC 104 [31]
zein-taqze R TACTGCATCCATGGGTTCAT
zein-taqze P AGCTTGATGGCGTGTCCGTCCCT
hmg hmg F TTGGACTAGAAATCTCGTGCTGA 79 [31]
hmg R GCTACATAGGGAGCCTTGTCCT
hmg P CAATCCACACAAACGCACGCGTA
zSSIIb zSSIIb F CGGTGGATGCTAAGGCTGATG 88 [32]
zSSIIb R AAAGGGCCAGGTTCATTATCCTC
zSSIIb P TAAGGAGCACTCGCCGCCGCATCTG
Adh1 Adh1 F CGTCGTTTCCCATCTCTTCCTCC 135 [31]
Adh1 R CCACTCCGAGACCCTCAGTC
Adh1 P AATCAGGGCTCATTTTCTCGCTCCTCA

Fig. 1

Schematic diagram of the fusion fragment and standard plasmid molecule pUC57-NK603 A: The fusion fragment of NK603 event-specific sequence and maize reference gene sequences. The NK603 event-specific sequence was marked with single underline, the spacer sequence was shaded, the Adh1 sequence was shown without marks, the zSSIIb sequence was marked with underlined dashed line, the hmg sequence was marked with double underline, and the zein sequence was shown in the box. B: Schematic diagram of the standard plasmid molecule pUC57-NK603"

Fig. 2

Hot plots of duplex ddPCR of NK603 event and different reference genes A: One-dimension (1-D) hot plots of NK603 event and zein-ENDO gene for NK603/zein-ENDO duplex ddPCR; B: 1-D hot plots of NK603 event and Adh1 gene for NK603/Adh1 duplex ddPCR; C: 1-D hot plots of NK603 event and hmg gene for NK603/hmg duplex ddPCR; D: 1-D hot plots of NK603 event and zSSIIb gene for NK603/zSSIIb duplex ddPCR; E: 1-D hot plots of NK603 event and zein-taq gene for NK603/zein-taq duplex ddPCR; F: Two-dimension (2-D) hot plot of NK603/zSSIIb duplex ddPCR at annealing temperature of 60℃"

Fig. 3

Measured copy number ratios of duplex ddPCR assays of NK603/hmg, NK603/zSSIIb and NK603/zein-taq at different annealing temperature"

Fig. 4

One-dimension hot map of NK603/zSSIIb duplex ddPCR for optimization of primer/probe concentration and measured copy number ratios of NK603 event and zSSIIb gene by NK603/zSSIIb duplex ddPCR A: 1-D hot plots of NK603 event of duplex ddPCR at different primer/probe concentrations; B: 1-D hot plots of zSSIIb gene of duplex ddPCR at different primer/probe concentrations; C: Measured copy number ratios of NK603 event to zSSIIb gene at different primer/probe concentrations. Sample order: 1-4: The primer/probe concentration of NK603 event was the same as that of zSSIIb gene, corresponding sequentially to 100/50, 200/100, 400/200, 800 nmol·L-1/400 nmol·L-1. 5-8: The primer/probe concentration of NK603 event was different from that of zSSIIb gene, corresponding sequentially to 400/200, 200 nmol·L-1/100 nmol·L-1; 200/100, 400 nmol·L-1/200 nmol·L-1; 400/200, 800 nmol·L-1/400 nmol·L-1; 800/400, 400 nmol·L-1/200 nmol·L-1"

Fig. 5

The dynamic range of NK603/zSSIIb duplex ddPCR"

Table 2

Transgenic DNA content in blind samples measured by duplex ddPCR and qPCR"

方法
Method		 样品
Sample		 质量分数
Mass fraction (%)		 转基因DNA含量 Transgenic DNA content (%) 平均值
Mean		 标准偏差
SD		 变异系数
RSD (%)
1 2 3
ddPCR S1 100 50.39 50.60 52.17 51.05 0.97 1.91
S2 10 4.95 4.74 4.79 4.82 0.11 2.28
S3 6 3.05 3.08 3.07 3.07 0.02 0.50
qPCR S1 100 51.17 57.72 57.76 55.55 3.79 6.83
S2 10 5.28 4.86 5.12 5.09 0.21 4.17
S3 6 3.17 3.69 3.92 3.59 0.38 10.68
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