Semi-quantitative Event Specific Detection of Bt Maize (Event MON 810) using Real-Time PCR with SYBR Green Ι Chemistry

Abstract

The objective is this work is to do semi-quantitative analysis for Bt maize (event MON 810) using conventional Polymerase Chain Reaction (PCR) and Real Time PCR (SYBR green chemistry) from the fresh maize samples chosen arbitrarily from various locations. The protocol for extraction of good quality genomic Deoxyribonucleic acid (DNA) was standardized without the use of Liquid nitrogen. The DNA used for further studies were obtained by using standardized protocol. The quality and concentration of Genomic DNA was checked through Agarose gel electrophoresis and taking absorbance at 260  and 280 nm.  Primer sets were designed specific to target gene (MON 810) and reference gene for further studies. Method validated for PCR reaction setup and cycling conditions for the amplification of desired target gene MON 810 of 170 bp and endogenous (zein) reference gene of 277 bp. on Real Time PCR and conventional PCR. Controls were used to avoid false positive or false negative results. The semi-quantitative analysis of GM content of selected maize seed samples was estimated by determining Cycle threshold (Ct) values of test and standard samples obtained in Real-Time PCR. The results obtained after analysis showed that almost 50% of the maize samples contain more than 0.1% GM content.