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Abstract Reference molecule is a recombinant plasmid which contain endogenous and exogenous genes. In order to provide a reference material for genetically modified organisms detection, the reference molecule which included six genes(Cauliflower mosaic virus(CaMV 35S)、Bacillus thuringiensis cry1Ab(cry1Ab)、sucrose phosphate synthase(SPS)、fiber-specific acyl carrier protein(fsACP)、nopaline synthase(NOS)、neomycin-3'-phosphotransferase (NPTⅡ) was constructed and confirmed by PCR, restriction analysis and sequencing. The repeatability, uniformity, homogeneity, stability and quantification method were also analyzed. The results shown as following: The quantitative accuracy of reference molecule which quantified by digital PCR was highest, followed by the PicoGreen method, and the quantitative accuracy of reference molecule which quantified by spectrophotometry was last; The repeatability, uniformity and homogeneity of reference molecules were very well, and the reference molecule could be used as the candidate of reference material; After one month stored at low temperature, the stability of reference molecule shown well. But the results of stability at high temperature shown that the reference molecule could not be stored. The reference molecule could be used for quantitative analysis of transgenic materials. The methods of construction, preparation and correctness verification of reference molecules can provide a reference example for the research of standard plasmid. Besides, this study provides a scientific basis and an example for the analysis and storage of reference molecules.
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Received: 25 February 2013
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