Development Triplex Real-Time PCR Screening Method to Detect GM Maize for ISO/IEC17025 Standard Accreditation
Keywords:Genetically Modified Corn; Screening Method; Multiplex Real Time PCR; Triplex Real Time PCR
Analysis of Genetically Modified plants in general laboratory uses qualitative PCR to detect CaMV 35S promoter and NOS terminator by single Real-Time PCR method. The quality of the DNA test was examined using plant specific reference gene, in the case of maize, the hmg gene was tested by PCR or Real-time PCR. This research has developed a method for screening genetically modified maize by testing three target genes simultaneously in a single reaction. The Triplex Real-Time PCR assay of CaMV 35S promoter, NOS terminator, and maize hmg gene were tested in the same reaction. Results show that the developed screening method was specific and sensitive for certified reference materials of corn-GM events. The Limit of detection was found at 0.01%. The concentration of the DNA template is 50-200 nanograms per reaction. Seventeen maize samples were also tested with this newly developed method. The test results were comparable to those of Single plex Real time PCR but this method was more efficient because it reduced both cost and time for the analysis It can be used as a standard screening method for maize and maize products. The method has already been approved as ISO / IEC17025 standard from Bureau of Laboratory Standards and used as a standard test method in laboratory of Department of Agriculture.
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