514 Chapter 29
nous reference genes, and donor organisms
(Mano et al. 2009 ). There are also other plat-
forms reported for high - throughput detection
based on multiplex assays on the basis of
SNPlex technology (Applied Biosystems),
which allows the simultaneous detection of
up to 79 SNPs in two panels containing 47
and 48 probes, respectively (Chaouachi et al.
2008 ).
While the quantifi cation obstacles for the
microarray approaches are surmounted, its
practical application in GMO analysis will
probably be restricted to an initial step for
detection and identifi cation that may be com-
bined with subsequent specifi c quantitative
approaches with validated real - time PCR
procedures.References
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D.C. : American Society for Microbiology.and two endogenous controls, the zein gene
for maize and lectin gene for soybean. A
previous step of multiplex PCR was used,
and one primer of each set was labeled. The
detection limit was below the EU recommen-
dation (0.25%) for each GMO. Xu et al.
(2006) designed three different microarrays,
the fi rst for the most used regulatory
sequences including the 35S promoter, 35S
terminator, nos promoter, nos terminator,
npt II terminator, and the FMV 35S promoter,
while the second microarray treated specifi c
gene inserts (soybean, cotton, and rapeseed),
and the third one was for endogenous con-
trols. A previous step of multiplex PCR was
done in which the amplifi ed fragments were
labeled with Cy5 - dCTP. The detection
ranged from 0.5% (soybean) to 1% (maize).
The same research group has also developed
a multiplex PCR - microarray for the detection
of GM soybean and six GM maizes. Amplifi ed
fragments were labeled with Cy5 - dCTP; the
limit of detection was similar to the other
microarray (0.5 – 1%) (Xu et al. 2007 ).
Leimanis et al. developed (2006, 2008) a
PCR - Microarray for the detection of fi ve
plant species and three screening sequences
(35S promoter, T - nos , and the npt II gene)
that has been validated (Leimanis et al.
2008 ). The detection was based on streptav-
idin - conjugated gold nanoparticles, and the
limit of detection was below 0.3%. Schmidt
et al. (2008) have developed a rapid multi-
plex PCR - microarray method for screening
GM canola. It contains probes that are con-
struct - specifi c to the 12 approved GM canola
lines in Canada, regulatory elements (CaMV
35S promoter, T - nos , and npt II), a canola -
specifi c endogenous gene, and endogenous
genes from heterologous crops. The limit of
detection was determined to range from 0.1%
to 0.5%. In addition, a real - time PCR array
has been developed for universal detection
and identifi cation of unapproved GMOs
that consists of 30 primer - probe sets distrib-
uted in a 96 - well plate for detection of GM
lines, recombinant DNA segments, endoge-