Identification and Quantification of Three Genetically Modified Insect Resistant Cotton Lines Using Conventional and TaqMan Real-Time Polymerase Chain Reaction Methods

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• 作者：Litao Yang ; Aihu Pan ; Kewei Zhang ; Jinchao Guo ; Changsong Yin ; Jianxiu Chen ; Cheng Huang ; and Dabing Zhang
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2005
• 出版时间：August 10, 2005
• 年：2005
• 卷：53
• 期：16
• 页码：6222 - 6229
• 全文大小：231K
• 年卷期：v.53,no.16(August 10, 2005)
• ISSN：1520-5118

文摘

As the genetically modified organisms (GMOs) labeling policies are issued in many countries,qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used forthe detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-timequantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences),which were approved for commercialization in China, were developed in this paper. Primer pairsspecific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and thespecific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531,GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. Inconventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531,GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR methodfor screening the three GM cottons was also established, which could save time and cost in practicaldetection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detectionof insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standardplasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c)and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitativePCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template,and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, allof the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton sampleswith 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of referencemolecule were reliable and practical for GM insect resistant cottons quantification. All of these resultsindicated that our established conventional and TaqMan real-time PCR assays were applicable todetect the three insect resistant cottons qualitatively and quantitatively.Keywords: Genetically modified organisms (GMOs); insect resistant cotton; multiplex PCR; real-timePCR; CpTI gene; Cry1A(c) gene