European Food Research and Technology

, Volume 230, Issue 2, pp 239–248 | Cite as

Development and in-house validation of a reference molecule pMIR604 for simplex and duplex event-specific identification and quantification of GM maize MIR604

Original Paper


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.


Duplex 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).


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, Bio-X Research Center, Key Laboratory of Genetics & Development and Neuropsychiatric Diseases, Ministry of Education, School of life Science and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.GMO Detection LaboratoryShanghai Entry–Exit Inspection and Quarantine BureauShanghaiPeople’s Republic of China

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