A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening.
The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.
Efficient detection strategies for genetically modified (GM) crops need to be in compliance with regulatory frameworks and address consumer concerns. The present review describes widely employed DNA-based technologies for GM detection. Polymerase chain reaction (PCR) and real-time PCR (qPCR) are the methods that can be used for qualitative and quantitative analysis of GM crops due to their specificity, sensitivity and robustness. With increase in number and complexity of genetic elements in newly developed GM events, strategies based on matrix approach, real-time PCR-based multi-target system, loop-mediated isothermal amplification, next generation sequencing, have emerged, which could facilitate cost-effective, rapid, on-site or high throughput GM detection.
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