Simplex and Duplex Event-Specific Analytical Methods for Functional Biotech Maize

GMO Testing Laboratory, Experiment Research Institute of National Agricultural Products Quality Management Service, Seoul, South Korea.
Journal of Agricultural and Food Chemistry (Impact Factor: 3.11). 09/2009; 57(16):7178-85. DOI: 10.1021/jf901078d
Source: PubMed

ABSTRACT Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: There are about 80 biotech crops' events which have been approved by safety assessment in Korea. They have been controlled by GMO (genetically modified organism) and LMO (living modified organism) labeling system. The DNA based detection method has been used as an efficient scientific management tool. Recently, the multiplex PCR and DNA chip have been developed as the simultaneous detection methods for several biotech crops' events. The event-specific multiplex PCR method was developed to detect biotech maize 5 events; MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed by 4 endogenous genes of soybean, maize, cotton, canola, and by introduced 2 elements & 7 genes; P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac, cry3B. The screening DNA chip was confirmed the specificity and 0.5% of sensitivity for 4 crops 12 events; soybean 1, maize 6, cotton 3 and canola 2. These multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis for biotech crops as the efficient detection methods by saving workload and time. Keywords: biotech crops; event-specific; multiplex PCR; screening; gene-specific; DNA chip.
    Journal of the Science of Food and Agriculture 11/2014; 94(14). DOI:10.1002/jsfa.6625 · 1.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Here, we present a simple mathematical algorithm which leads to a so called performance factor based on the amplification efficiency and the correlation coefficient. It is a single normalized number capable to characterize main amplification characteristics of single or multiplex quantitative real-time PCR systems. To show the usefulness of this performance factor, it was applied to calculate a validation criterion of individual quantitative PCR runs, to compare different real-time PCR systems and to compare premixed DNA-polymerase kits from various producers. KeywordsMultiplex-Real-time quantitative PCR-Comparing-Performance
    European Food Research and Technology 09/2010; 231(5):727-732. DOI:10.1007/s00217-010-1330-7 · 1.39 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250 bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs and the sensitivity threshold of the meqPCR was at least 50 ng 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.
    Analytical Biochemistry 07/2014; 464. DOI:10.1016/j.ab.2014.07.004 · 2.31 Impact Factor