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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (7): 949-955.doi: 10.3724/SP.J.1006.2018.00949

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

A Qualitative and Quantitative PCR Detection Method for Disease-resistant Genetically Modified Rice M12 and Its Derivates

Peng LI1,2,Lin ZHANG3,Ji-Ni YE4,Shi-Yao HE4,Jun-Wei JIA1,Ai-Hu PAN1,*(),Xue-Ming TANG1,*()   

  1. 1 Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences / Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China
    2 School of Life Science, Fudan University, Shanghai 200433, China
    3 Laiwu Vocational and Technical College, Laiwu 200433, Shandong, China
    4 School of Life Science, Taizhou University, Taizhou 318000, Zhejiang, China
  • Received:2017-12-03 Accepted:2018-03-26 Online:2018-07-10 Published:2018-04-16
  • Contact: Ai-Hu PAN,Xue-Ming TANG E-mail:aihup0318@163.com;saas_xmtang@foxmail.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31500461), the Key Technologies Program of Shanghai Agricultural Commission [2015(4-3)], SAAS Program for Excellent Research Team [2017(B-07)], and the Agricultural GMO Safety Supervision Program of Shanghai.

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Abstract:

In this study, the specific sequence of genetically modified Rice M12 and the endogenous reference gene sps were amplified to construct a T vector as the plasmid pM12 for establishing the qualitative and quantitative PCR detection method of transgenic Rice M12 and its derivates. The qualitative PCR method could specifically quantify the samples of M12 with the detection sensitivity about 100 copies of the rice haploid genome. On the basis of SYBR Green qPCR assay, R 2 values of standard curves of M12 and sps were 0.998 and 0.997, the amplification efficiency was 95.3% and 108.4%, respectively. Moreover, the standard deviations (SD) of repeatability ranged from 0.043 to 0.276. The limit of quantification (LOQ) and limit of detection (LOD) were 100 and 10 copies, respectively. The mixed rice sample containing 1.0% gene transforming into rice was exactly quantified by the developed quantitative PCR method, and the quantified bias between the true value and tested value was below 8.0%. In conclusion, these methods can be used for identifying and quantifying M12 and its derivatives.

Key words: event-specific, qualitative PCR, quantitative PCR, M12

Table 1

Primers used in qualitative (quantitative) PCR systems"

引物
Primer		 引物序列
Primer sequence (5°-3°)
sps-F TTGCGCCTGAACGGATAT [13]
sps-R CGGTTGATCTTTTCGGGATG [13]
M12-SF GTTGGAGATTTTGGGCTTG [2]
M12-SR ATAGCCTCTCCACCCAAGCG [2]
M12-F GTTGGAGATTTTGGGCTTGC
M12-R CGGCAAGAAACCATCCAGTT

Fig. 1

Specific sequence of genetically modified rice M12The arrows show the location and orientation of primers."

Fig. 2

PCR result of pM12M: DNA marker (DL1000); 1-2: primer: M12-F/R; 3-4: primer: sps-F/R; 5: negative control."

Fig. 3

Event-specific detection of qualitative PCR for M12: The qualitative PCR of event-specific M12 (A) and rice endogenous gene sps (B). M: DNA marker (DL1000); 1: pM12; 2: rice variety M12; 3: rice variety Huayou 14; 4: rice variety Huahui 1; 5: rice variety Hanyou 3; 6: rice variety Hanhui 3T; 7: negative control."

Fig. 4

Sensitivity detection of qualitative PCR for M12 The qualitative PCR of event-specific M12 (A) and rice endogenous gene sps (B).M: DNA marker (DL1000); 1-5: 10 000, 1000, 100, 10, 5 copies of haploid rice genomic DNA; 6: negative control."

Table 2

Limits of detection and quantification (LOD and LOQ) of M12 gene quantitative PCR"

拷贝数
Copy		 出现次数/检测次数
Signal rate (positive signals)		 循环数平均值
Ct mean		 标准差
SD
10000000 9/9 6.54 0.256
1000000 9/9 9.72 0.324
100000 9/9 13.15 0.236
10000 9/9 17.53 0.103
1000 9/9 20.82 0.265
100 9/9 24.12 0.231
10 2/9

Fig. 5

Amplification plots and standard curves of M1210 000 000, 1 000 000, 100 000, 10 000, 1000, and 100 copies of plasmid DNA respectively"

Fig. 6

Amplification plots and standard curves of sps gene10 000 000, 1 000 000, 100 000, 10 000, 1000, and 100 copies of plasmid DNA, respectively."

Table 3

Repeatability of M12 and sps gene quantitative PCR assays"

质粒分子DNA拷贝数
Copies of plasmid DNA		 sps M12
循环数平均值
Ct mean		 循环数标准差
Ct SD		 循环数平均值
Ct mean		 循环数标准差
Ct SD
1000000 12.15 0.087 6.52 0.276
100000 15.30 0.076 9.72 0.154
10000 18.62 0.067 13.13 0.186
10000 21.91 0.043 17.52 0.133
1000 24.32 0.123 20.71 0.165
100 27.60 0.124 24.05 0.134

Table 4

Mixed sample analysis of M12 quantitative PCR assays"

混合样本
Mixed samples (%)		 平均值1
Mean 1		 平均值2
Mean 2		 平均值3
Mean 3		 平均值
Mean (%)		 标准差
SD
1.0 0.95 0.95 0.88 0.92 0.04
0 0 0 0 0 —
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