Article
Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing
Registro en:
LIANG, Chanjuan et al. Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing. Anal Bioanal Chem, n. 406, p. 2603-2611, 2014.
1618-2650
10.1007/s00216-014-7667-1
Autor
Liang, Chanjuan
van Dijk, Jerone
Scholtens, Ingrid M. J
Staats, Martija
Prins, Theo W
Voorhuijzen, Marleen M
Silva, Andrea M. da
Arisi, Ana Carolina Maisonnave
Dunnen, Johan T den
Kok, Esther J
Resumen
The growing number of biotech crops with novel
genetic elements increasingly complicates the detection of
genetically modified organisms (GMOs) in food and feed
samples using conventional screening methods. Unauthorized
GMOs (UGMOs) in food and feed are currently identified
through combining GMO element screening with sequencing
the DNA flanking these elements. In this study, a specific and
sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the
recently marketed MIR162 maize and COT102 cotton. Furthermore,
SiteFinding-PCR in combination with Sanger,
Illumina or Pacific BioSciences (PacBio) sequencing was
performed targeting the flanking DNA of the vip3Aa20 element
in MIR162. De novo assembly and Basic Local Alignment
Search Tool searches were used to mimic UGMO identification.
PacBio data resulted in relatively long contigs in the
upstream (1,326 nucleotides (nt); 95 % identity) and downstream
(1,135 nt; 92 % identity) regions, whereas Illumina
data resulted in two smaller contigs of 858 and 1,038 nt with
higher sequence identity (>99 % identity). Both approaches
outperformed Sanger sequencing, underlining the potential for
next-generation sequencing in UGMO identification.