Event-specific Quantitative Polymerase Chain Reaction Research Articles

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3'-junction of the insertion site.

MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3'-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Event-specific qualitative and quantitative polymerase chain reaction methods for detection of insect-resistant genetically modified Chinese cabbage based on the 3′-junction of the insertion site

Transgenic Chinese cabbage 416-3 was developed in Korea by a transformation event involving modified insectresistant gene (cry1Ac1). To monitor unintended release of genetically modified (GM) Chinese cabbage in the future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of GM cabbage is requisite. To develop qualitative and quantitative polymerase chain reaction methods for the insect-resistant GM Chinese cabbage, a cytosolic glutathione reductase (BcgGR1) gene was used as the endogenous reference gene. Primer pairs CGR-1/-2, amplifying the Chinese cabbage endogenous gene, yielded an expected amplicon of 121 bp, whereas no amplified product was observed when DNA samples from seven non-cabbage plants were used as templates. The event-specific primer pairs amplifying the junction site between the endogenous genome sequence and the transferred DNA of GM event 416-3, produced amplicons of desired size by qualitative polymerase chain reaction (PCR) assay. An eventspecific quantitative PCR detection method was established using a TaqMan probe and a standard plasmid as a reference molecule, which contained both endogenous and event-specific sequences. For the validation of this method, three different compositions of w/w mixed samples (containing transgenic DNA at 5, 1, and 0.5% of total DNA in the control samples) were quantified. The precision, expressed as standard deviation (SD) and relative standard deviations (RSD), deviated by 0.03–0.26% and 4.75–8.06%, respectively. These results clearly demonstrate that the PCR methods developed herein can be used for event-specific qualitative and quantitative testings of insect-resistant GM Chinese cabbage.