Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2013, Vol. 39 ›› Issue (07): 1141-1147.doi: 10.3724/SP.J.1006.2013.01141

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

Event-Specific PCR Detection Method of Genetically Modified Maize MIR162 and Its Standardization

ZHANG Guang-Yuan1,2,SUN Hong-Wei1,LI Fan1,YANG Shu-Ke1,LU Xing-Bo1,*,ZHAO Lei2,*   

  1. 1 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Plant Virology, Jinan 250100, China; 2 College of Life Science, Shandong Normal University, Jinan 250014, China
  • Received:2012-12-27 Revised:2013-02-15 Online:2013-07-12 Published:2013-04-23
  • Contact: 路兴波, E-mail: luxb99@sina.com, Tel: 0531-83179095; 赵蕾, E-mail: zhaolei@sdu.edu.cn, Tel: 0531-88177190

Abstract:

Genetically modified maize MIR162 with resistances to Lepidoptera insects is developed by U.S. Syngenta Company through recombinant DNA technology. The purpose of this study was to establish and standardize the event-specific detection method of this line in China. The primers and Taqman probe were designed based upon the revealed 3' and 5' flanking sequences of MIR162. After validated by six external laboratories, results showed that the qualitative PCR detection method could specifically detect the samples of MIR162 with the detection sensitivity about 0.1%, a good repeatability and reproducibility. The quantitative PCR system was based on TaqMan. Two standard curves and corresponding linear regression equations of Quantitation-Ct between endogenous zSSIIb gene and transgenic target gene of MIR162 maize in standard material were generated. Results showed that the standard curves had good linear relationships. Their R2 values both were 0.999 and the recovery rates were 98.622% and 99.602%. Moreover, the standard deviations (SD) and relative standard deviations (RSD) of repeatability ranged from 0.014 to 0.209 and 0.049% to 0.879%. The limit of detection (LOD) was 0.01%. Thereafter, three mixed corn samples containing 1.0%, 0.5%, and 0.1% MIR162 were quantified employing the developed quantitative PCR method, and the quantified bias between the true value and tested value was below 8%. All these results suggested that the developed qualitative and quantitative PCR methods can be used for the identification and quantification of GM maize MIR162.

Key words: Event-speci?c, MIR162, Qualitative PCR, Taqman real-time PCR, Standardization

[1]Wei J-J(魏俊杰). Detection methods of transgenic products. J Hebei Agric Sci (河北农业科学), 2011, 15(11): 48–49 (in Chinese with English abstract)



[2]Forte V T, Di-Pinto A, Martino C, Tantillo G M, Grasso G, Schena F P. A general multiplex-PCR assay for the general detection of genetically modified sya and maize. Food Control, 2005, 16: 535–539



[3]Xu W-T(许文涛), Bai W-B(白卫滨), Luo Y-B(罗云波), Yuan Y-F(元延芳), Huang K-L(黄昆仑). Research progress in detection technique for genetically modified organisms. J Agric Biotechnol (农业生物技术学报), 2008, 16(4): 714–722 (in Chinese with English abstract)



[4]Gachet E, Martin G G, Vigneau F, Meyer G. Detection of genetically modified organism (GMOs) by PCR: A brief review of methodologies available. Trends Food Sci Technol, 1999, 9: 380–388



[5]Isabel T, Pieter W, Marc V, Anne M, Erik V B, Guy V E, Marc D L. Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176 and GA21 maize and transgenic GT73 canola. J Agric Food Chem, 2005, 53: 3041–3052



[6]Yang L T, Pan A H, Zhang K W, Yin C S, Qian B J, Chen J X, Huang C, Zhang D B. Qualitative and quantitative PCR methods for event-speci?c detection of genetically modi?ed cotton Mon1445 and Mon531. Transgenic Res, 2005, 14: 817–831



[7]Wu G, Wu Y H, Xiao L, Lu C M. Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235. Transgenic Res, 2008, 17: 851–862



[8]Pan A H, Yang L T, Xu S C, Yin C S, Zhang K W, Wang Z Y, Zhang D B. Event-speci?c qualitative and quantitative PCR detection of MON863 maize based upon the 3'-transgene integration sequence. J Cereal Sci, 2006, 43: 250–257



[9]Yang L T, Xu S C, Pan A H, Yin C S, Zhang K W, Wang Z Y, Zhou Z G, Zhang D B. Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312–9318



[10]Katarina C, Valérie C A, Marie-Noelle F, Kristina G, André K, Jana ?, Yves B. Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize. Anal Biochem, 2008, 376: 189–199



[11]Yang L T, Guo J C, Pan A H, Zhang H B, Zhang K W, Wang Z M, Zhang D B. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem, 2007, 55: 15–24



[12]Sissel B R, Marc V, Knut G B, Arne H J. Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays). Eur Food Res Technol, 2003, 216: 347-354



[13]Hari K S, Kae-Kang H, Wang S J, Liu L F, Chang M C. Simultaneous detection of eight genetically modified maize lines using a combination of event and construct specific Multiplex-PCR technique. J Agric Food Chem, 2008, 56: 8962–8968



[14]Tigst D, Indira R. Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event. Food Control, 2008, 19: 893–897



[15]Li X, Shen K L, Yang L T, Wang S, Pan L W, Zhang D B. Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 5' and 3' flanking sequences. Food Control, 2010, 21: 927–934



[16]Rott M E, Lawrence T S, Wall E M. Development of real-time PCR systems based on SYBR Green I amplifluore and Taqman technologies for specific quantitative detection of of the transgenic maize event GA21. Cereal Sci, 2004, 39: 99–107



[17]Jiang G-Z(姜国忠), Xie H(谢华), Guo Y-Z(郭玉忠), Fan T-L(范天黎), Xue L-X(薛乐勋). The comparison between two techniques in isolating 3' flanking region of Dunaliella salina actin gene. J Zhengzhou Univ (Med Sci)(郑州大学学报?医学版), 2004, 39(1): 41–44 (in Chinese with English abstract)

[1] Peng LI,Lin ZHANG,Ji-Ni YE,Shi-Yao HE,Jun-Wei JIA,Ai-Hu PAN,Xue-Ming TANG. A Qualitative and Quantitative PCR Detection Method for Disease-resistant Genetically Modified Rice M12 and Its Derivates [J]. Acta Agronomica Sinica, 2018, 44(7): 949-955.
[2] LI Jun,LIU Xin,CAO Ying-Long,WU Yu-Hua,LI Jian-Meng,WU Gang,ZHANG Li,LU Chang-Ming. Establishment of Phytase-Specific Qualitative PCR Detection Method and Construction of a Positive Plasmid Molecule [J]. Acta Agron Sin, 2012, 38(04): 639-647.
[3] YUAN Lei,SUN Hong-Wei,ZHAO Lei,YANG Chong-Liang,SHANG You-Fen,LU Xing-Bo. Analysis of Junction Sequence in the Transgenic Maize MON8817 and the Methods of Qualitative PCR Detction [J]. Acta Agron Sin, 2010, 36(2): 361-364.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!