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Establishment of quantitative analysis method for genetically modified maize using a reference plasmid and novel primers.

ABSTRACT: For the quantitative analysis of genetically modified (GM) maize in processed foods, primer sets and probes based on the 35S promoter (p35S), nopaline synthase terminator (tNOS), p35S-hsp70 intron, and zSSIIb gene encoding starch synthase II for intrinsic control were designed. Polymerase chain reaction (PCR) products (80~101 bp) were specifically amplified and the primer sets targeting the smaller regions (80 or 81 bp) were more sensitive than those targeting the larger regions (94 or 101 bp). Particularly, the primer set 35F1-R1 for p35S targeting 81 bp of sequence was even more sensitive than that targeting 101 bp of sequence by a 3-log scale. The target DNA fragments were also specifically amplified from all GM labeled food samples except for one item we tested when 35F1-R1 primer set was applied. A reference plasmid pGMmaize (3 kb) including the smaller PCR products for p35S, tNOS, p35S-hsp70 intron, and the zSSIIb gene was constructed for real-time PCR (RT-PCR). The linearity of standard curves was confirmed by using diluents ranging from 2×10(1)~10(5) copies of pGMmaize and the R(2) values ranged from 0.999~1.000. In the RT-PCR, the detection limit using the novel primer/probe sets was 5 pg of genomic DNA from MON810 line indicating that the primer sets targeting the smaller regions (80 or 81 bp) could be used for highly sensitive detection of foreign DNA fragments from GM maize in processed foods.

SUBMITTER: Moon GS

PROVIDER: S-EPMC3866725 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Establishment of quantitative analysis method for genetically modified maize using a reference plasmid and novel primers.

Moon Gi-Seong GS Shin Weon-Sun WS

Preventive nutrition and food science 20121201 4

For the quantitative analysis of genetically modified (GM) maize in processed foods, primer sets and probes based on the 35S promoter (p35S), nopaline synthase terminator (tNOS), p35S-hsp70 intron, and zSSIIb gene encoding starch synthase II for intrinsic control were designed. Polymerase chain reaction (PCR) products (80~101 bp) were specifically amplified and the primer sets targeting the smaller regions (80 or 81 bp) were more sensitive than those targeting the larger regions (94 or 101 bp). ...[more]

PMID: 24471096

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Project description:This study was conducted to follow up on apparent differences in growth, relative organ sizes, cellular stress and immune function in Atlantic salmon fed genetically modified (GM) Bt-maize compared to the non-modified parental maize line. Gene expression profiling on the distal intestinal segment and liver were performed by the cGRASP 16K salmonid cDNA microarray. Analysis of the intestinal microarray data revealed nearly 500 differentially regulated genes with a false discovery rate of zero, despite modest fold differences (average 2.4 for the down-regulated and 1.7 for the up-regulated genes). However, nine out of ten genes were found not to be significantly different when followed up by quantitative polymerase chain reaction (qPCR), but direction and magnitude of the change was generally confirmed, at least for the up-regulated genes. For the liver microarray data, statistical analyses could not be performed due to constraints in the design. qPCR revealed some differentially regulated genes in the liver, including up-regulation of gelsolin precursor, down-regulation of ferritin heavy subunit and a tendency towards down-regulation of metallothionein B. This, combined with up-regulation of anti-apoptotic protein NR13 and a tendency towards up-regulation of both ferritin heavy chain and metallothionein A and B in the distal intestine, suggests changes in cellular stress/ anti-oxidant status. This corresponds well with, and strengthens previous findings in these fish. To exclude possible confounding factors, the maize ingredients were analyzed for mycotoxins and metabolites, and the GM maize variety contain 90M-NM-<g/kg deoxynivalenol (DON), compared to levels below the detection limit in the non-GM maize. Numerous differences were also seen in the metabolite profiles of the two maize varieties, some of which seem to be connected to the mycotoxin level. The effects on salmon observed in the current and in previous studies correspond relatively well with effects of DON as reported in the literature for other production animals, but knowledge regarding effects and harmful dose levels in fish are practically non-existing, thus it is difficult to conclude whether the observed effects in the fish are caused by the DON level or by some other aspect of the GM maize ingredient.

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Establishment of quantitative analysis method for genetically modified maize using a reference plasmid and novel primers.

Publications

Establishment of quantitative analysis method for genetically modified maize using a reference plasmid and novel primers.

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