Development and in-house validation of a reference molecule pMIR604 for simplex and duplex event-specific identification and quantification of GM maize MIR604
Effective analysis methods for genetically modified organisms (GMOs) using reliable reference molecules as calibrators are necessary for the implementation of labeling policies. While no available detection systems based on the reference molecule have been reported for GM maize MIR604. Here we established the simplex and duplex qualitative and quantitative PCR systems for maize MIR604 employing a reliable new reference molecule pMIR604 as a calibrator, which contains fragments of the revealed 5′ flanking sequence of maize MIR604 and taxon-specific sequence zSSIIb. The limits of detection (LODs) were both 10 copies. The absolute LOD and limit of quantification were confirmed to be as low as 10 and 25 copies of pMIR604 through in-house validation both in simplex and duplex real-time PCR assays. For quantification of practical samples, results from five operators indicated that the biases of the data derived from each participant ranged from 2.00 to 23.00% and from 3.00 to 24.00% in simplex and duplex PCR systems, respectively. The relative standard deviations of the mean values for different GM content maize samples were all within 13.60% both in simplex and duplex analyses. This study showed that the detection systems of GM maize MIR604 using a new reference molecule pMIR604 as a calibrator are applicable for analysis of GM maize MIR604 and suitable for use as a preferable substitute of the reference material derived from plant raw materials.
KeywordsDuplex PCR Event specific Genetically modified organism MIR604 Reference molecule
This work was supported by the National Key Basic Research Program (2007CB109201), the National Transgenic Plant Special Fund (2008ZX08012-002, 003), the National Natural Science Foundation of China (30725022, 30700499), the national hightech project “863” (2006AA10Z443), the national “973” project (2007FY230100), Shanghai Municipal Committee of Science and Technology (08DZ0504300), and the Science and Technology program of Shanghai Agricultural committee (No. 10-2, 2006).
- 1.James C (2008) Global status of commercialize biotech/GM crops. ISAAA Briefs 39Google Scholar
- 3.Holst-Jensen A, Rønning SB, Løvseth A, Berdal KG (2003) PCR technology for screening and quantification. Anal Bioanal Chem 375:985–993Google Scholar
- 4.Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N (2002) Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int 85:1077–1089Google Scholar
- 10.Corbisier P, Broeders S, Charels D, Trapmann S, Vincent S, Emons H (2007) Certification of plasmidic DNA containing MON 810 maize DNA fragments, ERM®-AD413. European Commission, Joint Research Centre, Institute for Reference Materials and MeasurementsGoogle Scholar
- 12.Savini C, Mones W, Querci M, Mazzara M, Cordeil S, Van den Eede G, Zhang D (2007) Event-specific method for the quantification of maize line MIR604 using real-time PCR protocol. European Commission, Joint Research Centre, Institute for Health and Consumer Protection Biotechnology & GMOs UnitsGoogle Scholar
- 14.Ministry of Agriculture of People’s Republic of China (2007) Bulletin No. 953 and 869 of Ministry of AgricultureGoogle Scholar
- 17.Trapmann S, Conneely P, Contreras M, Corbisier P, Gioria S, Van-Nyen M, Vincent S, Emonsn H (2006) The certification of reference materials of dry-mixed maize powder with different mass fractions of MIR604 maize. European Commission, Joint Research Centre, Institute for Reference Materials and MeasurementsGoogle Scholar
- 18.Zhang D, Corlet A, Fouilloux S (2007) Impact of genetic structures on haploid genomes based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications. Transgenic Res 17:393–402CrossRefGoogle Scholar
- 19.Savini C, Mones W, Querci M, Mazzara M, Cordeil S, Van den Eede G, Zhang D (2007) Event-specific method for the quantification of maize line MIR604 using real-time PCR protocol. EU-CRLGoogle Scholar