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| Event-specific PCR Detection Methods of Genetically Modified Cotton (Gossypium hirsutum) MON757 and Their Application |
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Abstract Genetically modified cotton (Gossypium hirsutum) event MON757 expressing Cry1Ac protein was developed by U.S. Monsanto Company targeting Lepidoptera pests. It has already been approved for planting or using for food/feed in the United States, Canada, Australia and other countries, but has not yet been permitted for planting or application in China. At present, there are not specific methods for Genetically Modified (GM) cotton MON757 reported to identify the homozygous/heterozygous states or to quantify its content in mixed samples. In order to detect GM cotton MON757 in commercial cotton seeds of China, we established the specific homozygous/heterozygous qualitative PCR method and the event-specific quantitative Real-time PCR (qRT-PCR) method for MON757 based on the DNA sequences of the junctions between exogenous DNA fragments and cotton genomic DNA. The homozygous/heterozygous qualitative PCR method using 3 primers respectively located in the exogenous DNA segments and the cotton genomic DNA sequence on both sides of the insertion fragments could accurately identify homozygous and heterozygous states of MON757. The qRT-PCR method established in this study could specifically detect the samples of MON757 with the limit of detection (LOD) about 11 copies and the limit of quantitative (LOQ) about 44 copies. We tested 49 samples of cotton seeds from the market in Hubei province of China, and 5 samples were identified having MON757. In order to quantify the MON757 content of those 5 samples, 600 seeds of each sample were mixed for quantitative test using event-specific qRT-PCR method. At the same time, we tested 60 seeds from each samples using the homozygous/heterozygous qualitative PCR method to calculate the MON757 content. The results of both methods showed that the content of MON757 of one sample was about 1.5% and other 4 samples are all over 20%. The homozygous/heterozygous PCR method and the qRT-PCR method for MON757 that we developed are suitable and useful for homozygous and heterozygous states identification of cotton plants and seeds in the field and for quantifying the content MON757 from mixed samples.
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Received: 16 December 2015
Published: 06 May 2016
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