Detection and quantification of transgenes in grains by multiplex and real-time PCR.
Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), CONICET, Fundación M Lillo, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.
Hugo R
HR
Permingeat
Martín I
MI
Reggiardo
Rubén H
RH
Vallejos
D000595:Amino Acid Sequence; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001427:Bacterial Toxins; D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000032:analysis*; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D019076:Transgenes; D003313:Zea mays / Q000235:genetics*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D018744:DNA, Plant; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis