Development of an event-specific Real-time PCR detection method for the transgenic Bt rice line KMD1 |
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Authors: | Ruzha Babekova Tristan Funk Sven Pecoraro Karl-Heinz Engel Ulrich Busch |
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Affiliation: | 1.Lehrstuhl für Allgemeine Lebensmitteltechnologie,Technische Universit?t München,Freising-Weihenstephan,Germany;2.Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit,Oberschleissheim,Germany |
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Abstract: | The present study describes an event-specific quantitative Real-time PCR detection method for the transgenic Bt rice line
Kemingdao 1 (KMD1). This rice line which is not approved in any country so far is likely to be approved in China in the near
future. The developed primers amplify a DNA sequence spanning the integration site of the genetic construct in KMD1. DNA sequence
information of this unknown site necessary for primer design was achieved using SiteFinding-PCR technique. The specificity
of the detection method was shown by testing a number of different transgenic and conventional plant varieties (e.g. rice
LL 601, LL 62, Bt 63). As alternative to genomic DNA, we developed double target hybrid amplicons as synthetic calibration
standards in Real-time PCR. These amplicons contained both one copy of the KMD1 event-specific sequence and one copy of a
sequence of the rice reference gene gos9. The limit of quantification (LOQ) of the method was tested to be 0.05%.
R. Babekova and T. Funk contributed equally to this paper. |
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Keywords: | GMO Transgenic rice Kemingdao 1 (KMD1) Event-specific Real-time PCR SiteFinding-PCR Hybrid amplicon |
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