Development of Quantitative Duplex Real-Time PCR Method for Screening Analysis of Genetically Modified Maize

Abstract

A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstu#s and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

Interlaboratory Validation of Quantitative Duplex Real-Time PCR Method for Screening Analysis of Genetically Modified Maize

“…The levels of obtained LOQ, trueness and precision were almost the same as those of other established methods 9), 10) and the single laboratory evaluation 5) ῌ We thus consider that the duplex real-time PCR a good candidate for routine screening for GM maize commingled in agricultural crops.…”

“…To determine the Cf value for P35S, we used MON810 as a representative of GM maize both because it has been widely used, and because it has only one P35S segment per GM haploid, as the previous single laboratory evaluation described 5) ῌ The Cf values for GA21 and P 35S were measured independently with two real-time PCR instruments, the AB 7900 and AB 7500. The Cf values determined are listed in Table 1.…”

“…The standard plasmid pSCM which contains the specific sequence fragments from GA21, P35S and SSIIb, was prepared according to the previous report 5) and used as the calibrator for the quantification.…”

“…In this regard, screening methods are highly cost-and time-e#ective for routine monitoring. The Ministry of Health, Labour and Welfare (MHLW) of Japan announced a screening method combining the quantification of a P35S region and the constructspecific quantification of GA21 maize, which has been o$cially used as a quantitative screening method for GM maize῏ 3 ῌ To further pursue more convenient and e$cient methodology, we developed a duplex real-time PCR method which simultaneously quantifies the P35S region and an event-specific segment of GA21 5) ῌ The developed duplex screening method will reduce both the cost and time requirement of routine GMO analysis by half compared to the current screening method. These quantitative methods are based on a real-time PCR technique for relative quantification between target and taxon-specific sequences.…”

Research activities for the monitoring of genetically modified organisms in Japan

“…To improve the efficiency of the screening procedure, we developed a new screening method utilizing duplex real-time PCR, in which the sequences of p35S and the event-specific sequence of GA21 were simultaneously quantified [11]. As an efficient alternative to the existing screening method, we chose p35S and an event-specific segment for GA21 as the targets of this method.…”