Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (7): 146-152.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1330
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CAO Ying-fang1,2(), ZHAO Xin2, LIU Shuang2, LI Rui-huan2, LIU Na2, XU Shi-yong2, GAO Fang-rui2, MA Hui3, LAN Qing-kuo2, TAN Jian-xin1, WANG Yong1,2()
Received:
2021-10-20
Online:
2022-07-26
Published:
2022-08-09
Contact:
WANG Yong
E-mail:1336672682@qq.com;wytaas@126.com
CAO Ying-fang, ZHAO Xin, LIU Shuang, LI Rui-huan, LIU Na, XU Shi-yong, GAO Fang-rui, MA Hui, LAN Qing-kuo, TAN Jian-xin, WANG Yong. Establishment of Real-time Fluorescent Quantitative PCR Detection Method for Genetically Modified Herbicide-tolerant Soybean GE-J12[J]. Biotechnology Bulletin, 2022, 38(7): 146-152.
基因名称 Gene name | 引物和探针序列 Primer and probe sequence | 扩增片段大小 Amplified fragment size/bp | 来源 Source |
---|---|---|---|
GE-J12转化体 GE-J12 transformant | 5'-GGCTTTACTAAAATATAAATCCTAA-3' | 91 | 自行设计 |
5'-GGCGTTAATTCAGTACATTA-3' | |||
5'-FAM-TGACGCTTAGACAACTTAATAACACAT-BHQ1-3' | |||
Lectin | 5'-GCCCTCTACTCCACCCCCA-3' | 118 | 农业部2031号公告-8-2013 |
5'-GCCCATCTGCAAGCCTTTTT-3' | |||
5'-FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1-3' |
Table 1 Primers and probes used in this study
基因名称 Gene name | 引物和探针序列 Primer and probe sequence | 扩增片段大小 Amplified fragment size/bp | 来源 Source |
---|---|---|---|
GE-J12转化体 GE-J12 transformant | 5'-GGCTTTACTAAAATATAAATCCTAA-3' | 91 | 自行设计 |
5'-GGCGTTAATTCAGTACATTA-3' | |||
5'-FAM-TGACGCTTAGACAACTTAATAACACAT-BHQ1-3' | |||
Lectin | 5'-GCCCTCTACTCCACCCCCA-3' | 118 | 农业部2031号公告-8-2013 |
5'-GCCCATCTGCAAGCCTTTTT-3' | |||
5'-FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1-3' |
Fig. 1 Specific detection results via real-time fluorescence quantitative PCR 1:Genetically modified soybean GE-J12. 2:Soybean recipient Jack,other genetically modified soybeans,genetically modified corn,genetically modified rapeseed,genetically modified rice,genetically modified cotton,and other non-transgenic soybean mixtures
Fig. 2 Optimization results of real-time fluorescent quanti-tative PCR system 0:Blank control;1:primer 0.2 µmol/L,and probe 0.1 µmol/L;2:primer 0.4 µmol/L,and probe 0.2 µmol/L;3:primer 0.5 µmol/L,and probe 0.25 µmol/L;4:primer 0.6 µmol/L,and probe 0.3 µmol/L;5:primer 0.8 µmol/L,and probe 0.4 µmol/L;6:primer 1.0 µmol/L,and probe 0.5 µmol/L
测试样品Testing sample | 重复1 Repeat 1 | 重复2 Repeat 2 | 重复3 Repeat 3 | 平均偏差 Mean deviation | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
拷贝数 Number of copies | 含量 Content/% | 拷贝数 Number of copies | 含量 Content/% | 拷贝数 Number of copies | 含量 Content/% | ||||||||
GE-J12 | Lectin | GE-J12 | Lectin | GE-J12 | Lectin | ||||||||
GE-J12(5%) | 336.20 | 6 322.67 | 5.32 | 318.27 | 6 496.33 | 4.90 | 333.47 | 6 660.00 | 5.01 | 2.87 | |||
GE-J12(3%) | 125.97 | 4 719.33 | 2.67 | 256.67 | 8 025.00 | 3.20 | 238.33 | 8 155.67 | 2.92 | 6.78 | |||
GE-J12(1%) | 117.60 | 9 451.00 | 1.24 | 53.11 | 4 722.33 | 1.12 | 51.87 | 4 538.67 | 1.14 | 16.67 |
Table 2 Results of sample setting
测试样品Testing sample | 重复1 Repeat 1 | 重复2 Repeat 2 | 重复3 Repeat 3 | 平均偏差 Mean deviation | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
拷贝数 Number of copies | 含量 Content/% | 拷贝数 Number of copies | 含量 Content/% | 拷贝数 Number of copies | 含量 Content/% | ||||||||
GE-J12 | Lectin | GE-J12 | Lectin | GE-J12 | Lectin | ||||||||
GE-J12(5%) | 336.20 | 6 322.67 | 5.32 | 318.27 | 6 496.33 | 4.90 | 333.47 | 6 660.00 | 5.01 | 2.87 | |||
GE-J12(3%) | 125.97 | 4 719.33 | 2.67 | 256.67 | 8 025.00 | 3.20 | 238.33 | 8 155.67 | 2.92 | 6.78 | |||
GE-J12(1%) | 117.60 | 9 451.00 | 1.24 | 53.11 | 4 722.33 | 1.12 | 51.87 | 4 538.67 | 1.14 | 16.67 |
DNA模板量 DNA template amount | 重复1 Repeat 1 | 重复2 Repeat 2 | 重复3 Repeat 3 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Ct mean | SD | RSD/% | Ct mean | SD | RSD/% | Ct mean | SD | RSD/% | ||||
100% | 24.13 | 0.05 | 0.23 | 24.09 | 0.07 | 0.31 | 23.92 | 0.08 | 0.35 | |||
20% | 26.29 | 0.07 | 0.26 | 26.24 | 0.03 | 0.12 | 26.19 | 0.07 | 0.26 | |||
4% | 28.51 | 0.04 | 0.15 | 28.47 | 0.06 | 0.23 | 28.57 | 0.15 | 0.51 | |||
0.8% | 30.73 | 0.06 | 0.20 | 30.69 | 0.06 | 0.20 | 30.75 | 0.18 | 0.59 | |||
0.16% | 32.89 | 0.14 | 0.43 | 32.96 | 0.20 | 0.59 | 33.11 | 0.16 | 0.48 | |||
0.032% | 35.21 | 0.42 | 1.20 | 35.22 | 0.37 | 1.04 | 35.44 | 0.67 | 1.89 | |||
0.006 4% | 37.32 | 1.04 | 2.79 | 36.94 | 0.86 | 2.33 | 37.67 | 0.99 | 2.62 | |||
Formula | Y=-3.158X+36.784 | Y=-3.132X+36.683 | Y=-3.286X+37.113 | |||||||||
R2 | 0.992 | 0.995 | 0.992 | |||||||||
Amplification efficiency | 107.3% | 108.6% | 101.5% |
Table 3 Sensitivity and repeatability test of real-time fluorescence quantitative PCR
DNA模板量 DNA template amount | 重复1 Repeat 1 | 重复2 Repeat 2 | 重复3 Repeat 3 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Ct mean | SD | RSD/% | Ct mean | SD | RSD/% | Ct mean | SD | RSD/% | ||||
100% | 24.13 | 0.05 | 0.23 | 24.09 | 0.07 | 0.31 | 23.92 | 0.08 | 0.35 | |||
20% | 26.29 | 0.07 | 0.26 | 26.24 | 0.03 | 0.12 | 26.19 | 0.07 | 0.26 | |||
4% | 28.51 | 0.04 | 0.15 | 28.47 | 0.06 | 0.23 | 28.57 | 0.15 | 0.51 | |||
0.8% | 30.73 | 0.06 | 0.20 | 30.69 | 0.06 | 0.20 | 30.75 | 0.18 | 0.59 | |||
0.16% | 32.89 | 0.14 | 0.43 | 32.96 | 0.20 | 0.59 | 33.11 | 0.16 | 0.48 | |||
0.032% | 35.21 | 0.42 | 1.20 | 35.22 | 0.37 | 1.04 | 35.44 | 0.67 | 1.89 | |||
0.006 4% | 37.32 | 1.04 | 2.79 | 36.94 | 0.86 | 2.33 | 37.67 | 0.99 | 2.62 | |||
Formula | Y=-3.158X+36.784 | Y=-3.132X+36.683 | Y=-3.286X+37.113 | |||||||||
R2 | 0.992 | 0.995 | 0.992 | |||||||||
Amplification efficiency | 107.3% | 108.6% | 101.5% |
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