Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (5): 1010-1016.doi: 10.3864/j.issn.0578-1752.2012.05.023

• RESEARCH NOTES • Previous Articles     Next Articles

Multiplex Tandem PCR Assays for the Detection of Genetically Modified Organisms

 WEI  Shuang, CHEN  Zhen, MA  Jun, BAI  Wei-Bin, WU  Xi-Yang   

  1. 1.暨南大学理工学院食品科学与工程系，广州 510632
    2.广东出入境检验检疫局检验检疫技术中心，广州 510623
  • Received:2011-11-03 Online:2012-03-01 Published:2011-12-29

PDF

742

Abstract: 【Objective】The objective of this study is to develop a multiplex tandem polymerase chain reaction (MT-PCR) gene disk method for the detection of genetically modified organism (GMO). 【Method】 The MT-PCR method was employed to detect NOS terminator, FMV35S promoter, alien genes including NPTⅡ, Cry1Ab, Cry1Ab/Ac, CP4-EPSPS and PAT, which was normally used in transformation, and endogenous genes such as IVR from maize, sad1 from cotton, and PEP from rapeseed meal. Inner and outer primers were specially designed for each gene. Following the first PCR reaction, the multiplexed amplicons were simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product was then diluted and analyzed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR used a template enriched in the amplicons of interest, the conditions could be optimized to significantly reduce ‘primer dimer’ formation allowing SYBR Green chemistry to be used for quantification. The final results were achieved by cycling curves and melting curve. 【Result】 The MT-PCR method was rapid (＜2 h), high-throughput (10 genes of each sample), sensitive (＞0.000292 ng), and specific in detecting GMO, such as cotton seeds, maize, rice and rapeseed meal. The method is making very fine distinctions about the three kinds of transgenic species can be made by this method and it fits for a large amount of detection. 【Conclusion】 The MT-PCR method is specific, stable and reliable, and will be an effective tool for the detection of GMO.

Key words: genetically modified organism (GMO), multiplex tandem polymerase chain reaction (MT-PCR), gene disk

[1] James D, Schmidt A M, Wall E, Green M, Masri S. Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis. Journal of Agricultural and Food Chemistry, 2003, 51(20): 5829-5834.

[2] Permingeat H R, Reggiardo M I, Vallejos R H. Detection and quantification of transgenes in grains by multiplex and real time PCR. Journal of Agricultural and Food Chemistry, 2002, 50(16): 4431-4436.

[3] Lam W Y, Yeung A C M, Tang J W, Ip M, Chan E W C, Hui M, Chan P K S. Rapid multiplex nested PCR for detection of respiratory viruses. Journal of Clinical Microbiology, 2007, 45(11): 3631-3640.

[4] Schmidt A, Sahota R, Pope D S, Lawrence T S, Belton M P, Rott M E. Detection of genetically modified canola using multiplex PCR coupled with oligo nucleotide microarray hybridization. Journal of Agricultural and Food Chemistry, 2008, 56(16): 6791-6800.

[5] Zhou P P, Zhang J Z, You Y H, Wu Y N. Detection of genetically modified crops by combination of multiplex PCR and low-density DNA microarray. Biomedical and Environmental Sciences, 2008, 21(1), 53-62.

[6] Zeitler R, Pietsch K, Waiblinger H U. Validation of real-time PCR methods for the quantification of transgenic contaminations in rape seed. European Food Research and Technology, 2002, 214(4): 346-351.

[7] Yamaguchi H, Sasaki K, Umetsu M. Two detection methods of genetically modified maize and the state of its import into Japan. Food Control, 2003, 14(3): 201-206.

[8] Germini A, Rossi S, Zanetti A, Corranini R, Fogher C, Marchelli R. Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food. Journal of Agricultural and Food Chemistry, 2005, 53(10): 3958-3962.

[9] 王  永, 兰青阔, 赵  新, 朱  珠, 程  奕. 转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用. 中国农业科学, 2009, 42(4): 1473-1477.

Wang Y, Lan Q K, Zhao X, Zhu Z, Cheng Y. Development and application of loop-mediated isothermal amplification for detection of genetically modified crops. Scientia Agricultura Sinica, 2009, 42(4): 1473-1477. (in Chinese)

[10] Fukuta S, Ohishi K, Yoshida K, Mizukami Y, Ishida A, Kanbe M. Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum. Journal of Virological Methods, 2004, 121(1): 49-55.

[11] Lee D, La Mura M, Allnutt T R, Powell W. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences. BMC Biotechnology, 2009, 9(1): 7.

[12] Ahmed F E. Detection of genetically modified organisms in foods. Trends in Biotechnology, 2002, 20(5): 215-223.

[13] Stanley K K. Multiplexed tandem PCR: Gene profiling from small amounts of RNA using SYBR Green detection. Nucleic Acids Research, 2005, 33(20): e180.

[14] Lau A, Sorrell T C, Chen S, Stanley K, Iredell J, Halliday C. Multiplex Tandem PCR: A novel platform for rapid detection and identification of fungal pathogens from blood culture specimens. Journal of Clinical Microbiology, 2008, 46(9): 3021-3027.

[15] Lau A, Sorrell T C, Lee O C, Stanley K, Halliday C. Colony multiplex tandem PCR for rapid, accurate identification of fungal cultures. Journal of Clinical Microbiology, 2008, 46(12): 4058-4060.

[16] Stark D, Al-Qassab S E, Barratt J L N, Stanley K, Roberts T, Marriott D, Harkness J, Ellis J T. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. Journal of Clinical Microbiology, 2010, 49(1): 257-262.

[17] Szewczuk E, Thapa K, Anninos T, McPhie K, Higgins G, Dwyer D E, Stanley K K, Iredell J R. Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness. BMC Infectious Diseases, 2010, 10(1): 113.

[18] 陈  贞, 芦春斌, 杨梦捷, 马  骏, 白卫斌, 吴希阳. 棉花中转基因成分多重PCR体系的建立. 中国农业大学学报, 2011, 16(3): 15-20.

Chen Z, Lu C B, Yang M J, Ma J, Bai W B, Wu X Y. Multiplex PCR for detection of genetically modified components in cotton seeds. Journal of China Agricultural University, 2011, 16(3): 15-20. (in Chinese)

[19] 中华人民共和国国家质量监督检验检疫总局. 转基因产品检测-核酸定性PCR检测方法. 北京: 中国标准出版社, 2004.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Detection of Genetically Modified Organisms and Derived Products-Qualitative PCR Methods Based on Nucleic Acid. Beijing: Standards Press of China, 2004. (in Chinese)

[20] 中华人民共和国国家质量监督检验检疫总局. 转基因产品检测-基因芯片检测方法. 北京: 中国标准出版社, 2004.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Detection of Genetically Modified Organisms and Genechip Detection Assay. Beijing: Standards Press of China, 2004. (in Chinese)

[21] 中华人民共和国国家质量监督检验检疫总局. 番茄中转基因成分定性PCR检测方法. 北京: 中国标准出版社, 2006.

General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. Method of Polymerase Chain Reaction for Detecting Genetically Modified Components in Tomato. Beijing: Standards Press of China, 2006. (in Chinese)

[22] Rnning S B, Vatilingom M, Berdal K G, Holst-Jensen A. Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays). European Food Research and Technology, 2003, 216(4): 347-354.

[23] Matsuoka T, Kuribara H, Takubo K, Akiyama H, Miura H, Goda Y, Kusakabe Y, Isshiki K, Toyoda M, Hino A. Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays). Journal of Agricultural and Food Chemistry, 2002, 50(7): 2100-2109.

[24] Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyada M. Novel reference molecules for quantitation of genetically modified maize and soybean. Journal of AOAC International, 2002, 85(5): 1077-1089.

[25] Mano J, Oguchi T, Akiyama H, Teshima R, Hino A, Furui S, Kitta K. Simultaneous detection of recombinant DNA segments introduced into genetically modified crops with multiplex ligase chain reaction coupled with multiplex polymerase chain reaction. Journal of Agricultural and Food Chemistry, 2009, 57(7): 2640-2646.
No related articles found!
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd