Abstract
Multiplex polymerase chain reaction (PCR) methods have been developed and validated for screening, tracing, and regulating genetically modified (GM) crops in quarantine and environmental monitoring. In this study, we aimed to develop a method to simultaneously detect four GM cotton varieties in order to establish a screening system for cotton volunteers. Based on the sequence of DNA in the junction between introduced gene and flanking genomic DNA of four GM cotton events, herbicide-tolerant MON88701 and DAS-81910-7 and insect-resistant COT102 and T304-40, event-specific primers were designed and a multiplex detection method was developed. The simplex PCR results supported the multiplex PCR results; the amplification efficiency of the novel multiplex PCR method was increased compared with that of the Joint Research Centre (JRC) method. Based on the accuracy and efficiency, the method can be applied to detect and identify randomly mixed reference materials and suspected cotton volunteers. To apply this multiplex PCR method to living modified (LM) environmental monitoring samples, we performed additional PCR analysis to identify whether the volunteers were the four LM cotton varieties. As a result, 66 cotton volunteers were identified with stack event, comprising one or two of the four LM cotton events, and all stacks have been approved in South Korea for food, feed, and processing. These results indicated that our novel multiplex method is suitable for LMO identification.
Highlights
Innovations in modern biotechnology have led to the development of genetically modified (GM) crops with herbicide tolerance (HT), insect resistance (IR), and quality and production improvements
Based on the simplex polymerase chain reaction (PCR) results, we developed a novel multiplex PCR method for the four living modified (LM) cotton varieties (Fig. 3)
To increase the amplification intensity of small PCR fragments, we used 4 pmol of primers for COT102 and 2 pmol for the other three LM cotton events in the multiplex PCR analysis; the amplification intensity was sufficient in the multiplex PCR
Summary
Innovations in modern biotechnology have led to the development of genetically modified (GM) crops with herbicide tolerance (HT), insect resistance (IR), and quality and production improvements. In 2017, over 67 countries adopted GM crops and 25 countries cultivated GM crops on 189.8 million hectares—an increase of 3% from 2016—for food, feed, and processing [1]. These GM crops have been expanded beyond maize, soybean, canola, and cotton, which are commercial crops, to include alfalfa, sugar beet, papaya, potato, and apple. Among the nucleic acid-based methods, PCR detection methods including simplex and multiplex PCRs are widely applied owing to their high sensitivity and accuracy [3]. They have been applied in GM crop detection for screening and quantifying GM crop-derived DNA. The application of these real-time PCR detection methods is limited as they require expensive laboratory equipment [8]; there is a need to develop conventional multiplex PCR detection methods for GMO identification
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