title: Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.
abstract: An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.
authors.affiliation: Agilent Technologies, RMZ Centennial, Bangalore 560048, India.
authors.forename: Supriya
authors.initials: S
authors.lastname: Patwardhan
authors.forename: Srikanth
authors.initials: S
authors.lastname: Dasari
authors.forename: Krishna
authors.initials: K
authors.lastname: Bhagavatula
authors.forename: Steffen
authors.initials: S
authors.lastname: Mueller
authors.forename: Saligrama Adavigowda
authors.initials: SA
authors.lastname: Deepak
authors.forename: Sudip
authors.initials: S
authors.lastname: Ghosh
authors.forename: Sanjay
authors.initials: S
authors.lastname: Basak
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods / Q000592:standards*; D004731:Endotoxins / Q000235:genetics* / Q000302:isolation & purification; D005504:Food Analysis / Q000295:instrumentation / Q000379:methods*; D059022:Food Safety; D003368:Gossypium / Q000235:genetics / Q000254:growth & development; D006460:Hemolysin Proteins / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D046208:Microchip Analytical Procedures; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D030821:Plants, Genetically Modified; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000235:genetics / Q000254:growth & development; D003313:Zea mays / Q000235:genetics / Q000254:growth & development
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis