Contents - Faperta

Detection and Monitoring of Food and Food Products 469
Hupfer et al. (2000) developed a method for quantifi cation of transgenic insect-resistant
Bt maize by single- and dual-competitive PCR. The analysis of mixtures of DNA solutions,
as well as of maize fl ours containing defi ned amounts of Bt maize, demonstrated the usefulness
of single-competitive PCR based on co-amplifi cation of the CDPK promoter/cryIAb
gene region of Bt maize and an internal standard. Upon heat treatment of DNA solutions
and maize fl our, the recovery of the Bt proportion compared to the starting material determined
by single-competitive PCR decreased signifi cantly. This systematic error could be
compensated for by using a dual-competitive approach based on PCR quantifi cation of the
transgenic target sequence of Bt maize and of the maize specifi c invertase gene.
Multiplex PCR-Based Detection Methods
Several target DNA sequences can be screened with multiplex PCR-based methods, and
detected in a single reaction. In the multiplex method, fewer reactions are needed to test a
sample for potential presence of transgenic plant-derived DNA. Development of multiplex
assays requires careful testing and validation. After the PCR, the resulting pool of amplifi
ed fragments needs to be analyzed further to distinguish between the amplicons.
Multiplex assay for detection of fi ve transgenic maize varieties (Bt11, Bt176, Mon810, T25,
and GA21) has been well standardized (Matsuoka et al., 2001).
Quantitative PCR
In principle, PCR-based quantifi cation can be performed either after completion of the
PCR (end-point analysis), or during the PCR (real-time analysis) (Table 16.2). Conventional
PCR measures the products of the PCR reaction at the end point in the reaction profi le.
End-point analyses are based on comparison of the fi nal amount of amplifi ed DNA of two
DNA targets, the one to be quantifi ed and a competitor (an artifi cially constructed DNA
that is added in a small and known quantity prior to the PCR amplifi cation, which is
co-amplifi ed with the target to be quantifi ed). The competitor has the same binding sites
for the same primer pair, but is of a different size. This is called competitive quantitative
PCR, and the two DNA targets are amplifi ed with equal effi ciency. A series of dilutions
TABLE 16.2
Methods for Quantifi cation of Derivatives in Genetically Modifi ed Food and Food Products
Method Genetically Modifi ed Material References
Double competitive PCR
methods
Real-time PCR methods Roundup Ready soybean
(event specifi c)
Synthetic cry1Ab gene
Roundup Ready soybean
(raw materials)
Van den Eede et al. (2000)
Bt176 maize (raw materials) Van den Eede et al. (2000)
P-35S (screening) Hardegger, Brodmann, and Herrmann (1999)
T-Nos (screening) Hardegger, Brodmann, and Herrmann (1999)
Epsps gene (RoundupReady) Studer et al. (1998)
Synthetic cry1Ab gene Studer et al. (1998)
Bt11 maize (event specifi c) Zimmermann, Luthy, and Pauli (2000)
Berdal and Holst-Jensen (2001), Terry and
Harris (2001), Taverniers et al. (2001)
Vaitilingom et al. (1999)