Event-specific multiplex PCR method for four genetically modified cotton varieties, and its application

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.

Development of a multiplex PCR method for identification of four genetically modified maize lines and its application in living modified organism identification

“…Recently, numerous high-throughput DNA-based detection methods have been developed and applied. Among them, the multiplex PCR method, which amplifies multiple DNA targets in one reaction, is a simple strategy for monitoring and screening multiple GM plant samples (Jo et al 2016;Shin et al 2016;Eum et al 2019).…”

“…The validation of GMO detection methods is very complex and depends on the availability, type, and quality of the template DNA. The quantity and quality of genomic DNA from GMO monitoring samples were not sufficient for analysis; we therefore used LOD and random CRM mixture analysis to verify the efficiency of the multiplex PCR method (Eum et al 2019). The analysis of serially diluted CRM template DNA revealed that the PCR bands were detected at a DNA concentration of 25 ng/µl (Fig.…”

Establishment and Application of a Monitoring Strategy for Living Modified Cotton in Natural Environments in South Korea

“…One oligonucleotide primer set for the adhC gene was used to confirm the stability of the PCR reactions. Specific PCR analysis using primer sets for 9 cotton events (T304-40, LLCOTTON25, MON1445, MON531, MON15985, MON88913, GHB614, GHB119, and 281-3006) was conducted to characterize the collected suspicious samples, as previously described (Table 1) [21][22][23][24]. The certified reference materials that served as controls in the PCR reactions were purchased from the American Oil Chemist's Society (AOSC; Urbana, IL, USA) and the Institute for Reference Materials and Measurements (IRMM; Geel, Belgium).…”

Monitoring of Infant Formula and Baby Food for the Pat and NOS Terminator of Genetically Modified Maize and Soybean by Real-time PCR in Iran

“…Resistance to herbicides is the main characteristic of GMCs, and 47% of the world has planting areas of herbicide-resistant corn, soy, cotton, and canola. The usage of these products can significantly save on pesticides and herbicides, making the environment less contaminated (11). There are many reports from Iran (12), Serbia (13), and Italy (14) that GMOs are present in various foods.…”