Real-time quantitative PCR with SYBR green I for living modified roundup ready soybean = Roundup ready soybean의 정량 검출에서 SYBR green I을 이용하는 real-time PCR 조건의 분석

Abstract

Concerns on the safety of living modified (LM) crops have led to mandatory-labeling legislation of LM crops in many countries including Korea. An real-time PCR method for quantification of LM Roundup Ready soybean (RRS) with the double-stranded DNA intercalating dye, SYBR Green I, is described. Pairs of primers that specifically PCR-amplify the targeted 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and le1 gene sequences were selected by means of analysis of melting-curve plot of PCR products. Then, the specificity and general performance of the selected primer pairs were further increased by selecting optimum primer concentration by means of serial analyses of combination of different concentrations of each primer in the selected primer pairs. Using 1, 2, and 5% RRS test samples and applying the real-time PCR with SYBR Green I, mean values deviated from true values by 5.5 to 25%. The precision of the real-time PCR with SYBR Green I was comparable with that of the real-time PCR with TaqMan chemistry, which is widely used for the quantification of LM crops today. The results suggest that the real-time PCR with SYBR Green I, which does not require the high-cost dye labelling step for the initial primer or probe design, could be an alternative method for the quantification of LM Crops.