Multitarget Real-Time PCR-Based System: Monitoring for Unauthorized Genetically Modified Events in India

Abstract

A multitarget TaqMan real-time PCR (RTi-PCR) based system was developed to monitor unauthorized genetically modified (GM) events in India. Most of the GM events included in this study are either authorized for commercial cultivation or field trials, which were indigenously developed or imported for research purposes. The developed system consists of a 96-well prespotted plate with lyophilized primers and probes, for simultaneous detection of 47 targets in duplicate, including 21 event-specific sequences, 5 construct regions, 15 for transgenic elements, and 6 taxon-specific targets for cotton, eggplant, maize, potato, rice, and soybean. Limit of detection (LOD) of assays ranged from 0.1 to 0.01% GM content for different targets. Applicability, robustness, and practical utility of the developed system were verified with stacked GM cotton event, powdered samples of proficiency testing and two unknown test samples. This user-friendly multitarget approach can be efficiently utilized for monitoring the unauthorized GM events in an Indian context.

Development of Gene-Specific Real-Time PCR Screening Method for Detection ofcp4-epspsGene in GM Crops

“…The LOD between 0.05 and 0.01 (ng/μl) DNA was detected for cry1A.105 and cry2ab2 when a serial dilution of 100 % CRM MON 89034 was targeted in real-time assays, which corresponds to 18 to 4 copies (14). Recently, different DNA-based detection technologies have been developed to evaluate the copy number of cp4-epsps gene in different crops (20,19,15). In a multi-target system which was developed for the simultaneous detection of 47 targets, the system was capable of detecting 42, 6, and 88 copies of the cp4-epsps gene in cotton MON 88913, cotton MON 71800, and soybean GTS 40-3-2, respectively where the sensitivity of detection was lower than the method used in the present research (19).…”

“…Recently, different DNA-based detection technologies have been developed to evaluate the copy number of cp4-epsps gene in different crops (20,19,15). In a multi-target system which was developed for the simultaneous detection of 47 targets, the system was capable of detecting 42, 6, and 88 copies of the cp4-epsps gene in cotton MON 88913, cotton MON 71800, and soybean GTS 40-3-2, respectively where the sensitivity of detection was lower than the method used in the present research (19). In another study, the lowest copy number which was detectable based on loop-mediated isothermal amplification method was 4 copies of cp4-epsps (20).…”

“…Also, PCR techniques were employed to detect 35 S, Nos and cp4-epsps genes in GM soybean and maize (18). A multi-target Taq Man real-time PCR was developed for simultaneous detection of different GM crops including cotton, eggplant, maize, potato, rice, and soybean (19). More recently, a detection method based on loop-mediated isothermal amplification was used to monitor insect and herbicide resistant crops some of which harbored the cp4-epsps gene (20).…”

Approaches to check unauthorized genetically modified events in supply chain: Challenges and solutions in the Indian context

“…Event‐specific qPCR assays for MON531 and MON15985 can be employed for checking AGM, whereas qPCR assay targeting ctp2‐cp4epsps can detect unapproved Roundup® Ready events. Multi‐target qPCR system to check for AGM and UGM in the Indian context was developed, which simultaneously detects 47 targets, including 21 GM events, 5 construct regions, 15 transgenic elements, and 6 taxon‐specific targets for brinjal, cotton, maize, potato, rice, and soybean 59 . Potential qPCR assays 11,27,28,32–34,37–39,41–43,45–51 that can be employed to check for UGM are summarized in Table 2.…”

“…Multitarget qPCR system to check for AGM and UGM in the Indian context was developed, which simultaneously detects 47 targets, including 21 GM events, 5 construct regions, 15 transgenic elements, and 6 taxon-specific targets for brinjal, cotton, maize, potato, rice, and soybean. 59 Potential qPCR assays 11,27,28,[32][33][34][37][38][39][41][42][43][45][46][47][48][49][50][51] that can be employed to check for UGM are summarized in Table 2.…”