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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (7): 1818-1828.doi: 10.3724/SP.J.1006.2023.23048

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

Event-specific PCR detection method of transgenic maize ND207 and its standardization

CHANG Li-Juan1(), LIANG Jing-Gang2,*(), SONG Jun1, LIU Wen-Juan1, FU Cheng-Ping3, DAI Xiao-Hang1, WANG Dong1, WEI Chao1, XIONG Mei1   

  1. 1Institute of Quality Standard and Testing Technology Research, Sichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China
    2Development Center of Science and Technology, MARA, Beijing 100176
    3Institute of Remote Sensing and Digital Agriculture, Sichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China
  • Received:2022-06-16 Accepted:2022-11-25 Online:2023-07-12 Published:2022-12-30
  • Contact: *E-mail: liangjingang@agri.gov.cn E-mail:277989595@126.com;liangjingang@agri.gov.cn
  • Supported by:
    The Project of Agricultural National and Industrial Standards in 2020(农质标函(2020)128号);The Natural Science Foundation of Sichuan Province(2022NSFSC0140);The Fund of “1+9” Science and Technology Project of Sichuan Academy of Agricultural Sciences——Advanced Technology for Biosafety(1+9KJGG006)

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Abstract:

Transgenic maize ND207 is an insect-resistant maize with mCry1Ab and mCry2Ab genes developed by China Agricultural University. The objective of this study is to develop the event-specific PCR detection method for ND207. The PCR amplification was performed according to the primers provided by the ND207 developer. The sequence of insertion site of ND207 was sequenced and obtained 262 bp of the 5'-flanking sequence, including 109 bp vector sequence and 153 bp maize genome sequence, 316 bp of the 3'-flanking sequence, including 76 bp vector sequence and 240 bp maize genome sequence. Fourteen primers were designed at both ends to form 25 primer pairs. The best primer pair at the 3' end was selected to optimize the PCR reaction system and reaction condition. The event-specific qualitative PCR detection method of ND207 was established and the PCR product size was 166 bp. After testing, the results showed that the detection limit of this method was 0.1%, equivalent to 20 copies of ND207 specific molecule fragment. Eight GMO safety testing institutions in China tested specificity, detection limit, reproducibility of the method, and the circular verification report revealed that the method met the requirement of the national standard method, which could be promoted and applied in the testing industry. The established event-specific qualitative PCR detection method of ND207 provides the technical support for the safety supervision of ND207 and its derivatives in China.

Key words: transgenic maize, ND207, flanking sequence, event-specific, qualitative PCR

Fig. 1

Exogenous insertion sequence map of transgenic maize ND207"

Table 1

Primer pairs for PCR amplification of transgenic maize ND207"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 片段大小
Amplicon size (bp)		 引物位置
Primer position
ND207 5F 5'-CGGTCGATGAACGTGAACAAG-3' 254 5'-端旁侧序列
5'-flanking sequence
ND207 5R 5'-CAGTACATTAAAAACGTCCGCAA-3'
ND207 3F 5'-GTTTTTATGATTAGAGTCCCGCAAT-3' 303 3'-端旁侧序列
3'-flanking sequence
ND207 3R 5'-CAGGATGGGCTTCATGTACTCC-3'

Table 2

Primer sequence used for PCR"

名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物位置
Primer position
ND207 5'-F1
ND207-5'-F2
ND207-5'-F3		 5'-ATGAACGTGAACAAGGTGAAGC-3'
5'-CGATGAACGTGAACAAGGTGAA-3'
5'-AGGTGAAGCTCTACGACGC-3'		 5'-端旁侧序列
5'-flanking sequence
ND207-5'-R1 5'-CCGCAATGTGTTATTAAGTTGTCTA-3'
ND207-5'-R2 5'-AACGTCCGCAATGTGTTATTAAGT-3'
ND207-5'-R3 5'-ATGTGTTATTAAGTTGTCTAAGCGT-3'
ND207-3'-F1 5'-TGATTAGAGTCCCGCAATTATACAT-3' 3'-端旁侧序列
3'-flanking sequence
ND207-3'-F2 5'-CGATAGAAAACAAAATATAGCGCG-3'
ND207-3'-F3 5'-AACAAAATATAGCGCGCAATC-3'
ND207-3'-F4 5'-TGATTAGAGTCCCGCAATTATAC-3'
ND207-3'-R1 5'-AGGTCCTCCCGGAACGC-3'
ND207-3'-R2 5'-GACGACGGCGGGAAGCT-3'
ND207-3'-R3 5'-GTGCGCCGACGAGACG-3'
ND207-3'-R4 5'-CGTGGACGGCCTTCATAG-3'

Table 3

Primer combinations used for PCR"

编号
Number		 正向引物
Forward primer		 反向引物
Reverse primer		 片段大小
Amplication size (bp)		 引物位置
Position
5'-1 ND207-5'-F1 ND207-5'-R1 235 5'-端旁侧序列
5'-flanking sequence
5'-2 ND207-5'-F1 ND207-5'-R2 240
5'-3 ND207-5'-F1 ND207-5'-R3 230
5'-4 ND207-5'-F2 ND207-5'-R1 237
5'-5 ND207-5'-F2 ND207-5'-R2 242
5'-6 ND207-5'-F2 ND207-5'-R3 232
5'-7 ND207-5'-F3 ND207-5'-R1 222
5'-8 ND207-5'-F3 ND207-5'-R2 227
5'-9 ND207-5'-F3 ND207-5'-R3 217
3'-1 ND207-3'-F1 ND207-3'-R1 278 3'-端旁侧序列
3'-flanking sequence
3'-2 ND207-3'-F2 ND207-3'-R1 245
3'-3 ND207-3'-F3 ND207-3'-R1 237
3'-4 ND207-3'-F4 ND207-3'-R1 278
3'-5 ND207-3'-F1 ND207-3'-R2 257
3'-6 ND207-3'-F2 ND207-3'-R2 224
3'-7 ND207-3'-F3 ND207-3'-R2 216
3'-8 ND207-3'-F4 ND207-3'-R2 257
3'-9 ND207-3'-F1 ND207-3'-R3 219
3'-10 ND207-3'-F2 ND207-3'-R3 186
3'-11 ND207-3'-F3 ND207-3'-R3 178
3'-12 ND207-3'-F4 ND207-3'-R3 219
3'-13 ND207-3'-F1 ND207-3'-R4 166
3'-14 ND207-3'-F2 ND207-3'-R4 133
3'-15 ND207-3'-F3 ND207-3'-R4 125
3'-16 ND207-3'-F4 ND207-3'-R4 166

Fig. 2

Sequence alignment between both 5'-and 3'-flanking sequences of transgenic maize ND207 and genome sequence in WT maize The identical nucleotide is shaded with black color, and the nucleotide shaded with blue color means the base differences of the compared sequence."

Fig. 3

Screening of the primers for ND207"

Fig. 4

Screening for the primers specificity 1: the positive control; 2: the negative control; 3: the blank; 4: transgenic corn mixture samples; 5: transgenic rice mixture samples; 6: transgenic rape mixture samples; 7: transgenic soybean mixture samples; 8: transgenic cotton mixture samples."

Fig. 5

Screening for the primers sensitivity"

Fig. 6

Test of PCR reaction temperature and primer concentration"

Table 4

PCR reaction system"

试剂
Reagent		 终浓度
Final concentration		 体积
Volume
水 Water —
10×PCR缓冲液 10×PCR buffer solution 1× 2.5 µL
25 mmol L-1氯化镁溶液 25 mmol L-1 MgCl solution 1.5 mmol L-1 1.5 µL
dNTPs混合溶液(各2.5 mmol L-1) dNTPs mixture (each 2.5 mmol L-1) 0.2 mmol L-1 2.0 µL
正向引物10 µmol L-1 ND207-F Forward primer 10 µmol L-1 ND207-F 0.4 µmol L-1 1.0 µL
反向引物10 µmol L-1 ND207-R Reverse primer 10 µmol L-1 ND207-R 0.4 µmol L-1 1.0 µL
Taq DNA聚合酶 Taq DNA polyase 0.025 U µL-1 —
25 mg L-1 DNA模板 25 mg L-1 DNA templet 2.0 mg L-1 2.0 µL
总体积 Total volume 25.0 µL

Fig. 7

Test of method sensitivity"

Fig. 8

Test of method detection limit"

Fig. 9

Test of method reproducibility"

Table 5

Cyclic verification for the event-specific PCR detection method for ND207"

样品名称
Sample name		 定性PCR检测结果
Detection results of qualitative PCR
阳性对照Positive control + + + + + +
阴性对照Negative control - - - - - -
1% ND207玉米 1% transgenic maize ND207 + + + + + +
其他转基因玉米混合样Transgenic corn mixture samples - - - - - -
转基因大豆混合样Transgenic soybean mixture samples - - - - - -
转基因水稻混合样Transgenic rice mixture samples - - - - - -
转基因棉花混合样Transgenic cotton mixture samples - - - - - -
转基因油菜混合样Transgenic rape mixture samples - - - - - -
非转基因玉米混合样Non-transgenic maize mixture samples - - - - - -
0.1% ND207玉米 0.1% transgenic maize ND207 + + + + + +
0.05% ND207玉米 0.05% transgenic maize ND207 - - - - - -
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