Development and Interlaboratory Validation of Quantitative Polymerase Chain Reaction Method for Screening Analysis of Genetically Modified Soybeans

Abstract

A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined RSDr values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.Key words screening; quantification; genetically modified; real-time polymerase chain reaction; soybeanIn the past few decades, a huge variety of genetically modified (GM) crops have been developed and cultivated worldwide.1) Several countries legislate the labeling systems of authorized GM crops, or their derived foods and feeds. To enforce the labeling system, it is indispensable to define a practical threshold level of GM content and to develop validated quantitative methods for the estimation of unintentional commingling of GM organisms (GMO). GM crops are generally classified in terms of event which is identified by what transgene was inserted, and where on the chromosome it was inserted. There are many GM events approved in Japan.2)The quantitative methods of a glyphosate-tolerant soybean (GTS) Roundup ready soy (RRS) and five GM maize events, i.e., Bt11, Bt176, GA21, MON810, and T25, using real-time polymerase chain reaction (PCR) were developed and validated by an interlaboratory collaborative study, 3) and adopted as Japanese standard methods. 4)For DNA-based detection of GM crops, screening tests are highly cost-and time-effective methods. The Ministry of Health, Labour and Welfare (MHLW) of Japan announced a combinational quantification of P35S and GA21 maize as a quantitative screening method for GM maize and it has been officially adopted.4) Among approved GM soybean events, RRS has been cultivated since in 2001, and the commercial cultivation of a glufosinate-tolerant soybean, A2704-12 and the second generation of GTS, MON89788, have recently started. To analyze these novel GM soybean events, we have developed individual event-specific quantitative methods. 5,6) A practical quantitative screening method for GM soybeans is needed because many food products derived from soybeans are listed for labeling and then frequently analyzed in Japan. 7)In this study, we first developed a new quantitative screening method for RRS, A2704-12 and MON89788. The developed method was evaluated with an interlaboratory collaborative study. MATeRIALS ANd MeTHOdSPlant Materials MON89788 and RRS seeds were kindly provid...

Development and Interlaboratory Validation of a Novel Reproducible Qualitative Method for GM Soybeans Using Comparative Cq-Based Analysis for the Revised Non-GMO Labeling System in Japan

“…During GM soybean screening, real-time PCR assays were performed to detect the endogenous gene encoding soybean lectin1 ( Le1 ) , and the 35S promoter of cauliflower mosaic virus (P35S) and MON89788 event-specific (MON89788es) sequences …”

“…The interlaboratory study was designed, and the results were analyzed as previously described. , Fifteen laboratories in Japan participated in this study. The experimental protocols and test samples were prepared and provided by the Food Research Institute, National Agriculture and Food Research Organization.…”

Rapid Screening Detection of Genetically Modified Crops by Loop-Mediated Isothermal Amplification with a Lateral Flow Dipstick

“…Meanwhile, the weight-based mixing samples were used for direct LAMP analyses. Two GM soybean samples were previously prepared consisting of (i) 0.5% RRS and 0.5% MON87701 14 and (ii) 0.5% RRS, 0.5% MON89788, and 0.5% A2704-12 22 and three GM maize samples, consisting of (iii) 0.5% MON88017 and 0.5% 3272, 14 (iv) 0.5% MON810 and 0.5% GA21, 23 and (v) 0.4% Bt11 and 0.2% GA21. 24 LAMP Assay.…”

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

“…In addition, Southern blot is impractical for examining large population of plants as it requires many laborious and time-consuming steps [12]. Real-time PCR methods for CN determination have also been developed in grapevine [13], citrus [14], maize [15][16][17], tomato [18,19], soybean [20,21], rice [22,23], wheat [23,24] and others. Real-time PCR shows certain technical advantages, such as rapidity, requirement of low-quantity DNA, high repeatability and reproducibility, and the possibility of applying statistical tests to the quantitative results [13].…”