Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR |
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Authors: | Patrick Guertler Ingrid Huber Sven Pecoraro and Ulrich Busch |
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Affiliation: | (1) Bavarian Health and Food Safety Authority, Veterin?rstr. 2, 85764 Oberschlei?heim, Germany |
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Abstract: | The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive
and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the
EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time
PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right
border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO
testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection
of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation
coefficient (R
2) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing
of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further,
35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of
35 samples, PCR products for Kefeng 6 DNA were observed. |
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