A calibrator plasmid for quantitative analysis of insect resistant maize (Yieldgard MON 810) Rajashekhar V. Ballari a , Asha Martin a , Lalitha R. Gowda b,⇑ a Food Safety and Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute, Mysore 570 020, India b Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India article info Article history: Received 24 September 2012 Received in revised form 2 January 2013 Accepted 18 February 2013 Available online 26 February 2013 Keywords: Genetically modified organisms MON 810 maize Real-time PCR Reference materials Plasmid DNA calibrants Genomic DNA calibrants abstract Real-time PCR (RT-PCR) is the preferred method for the quantification of genetically modified organisms (GMOs) and implementation of labeling regulations. The precision, sensitivity, and reproducibility of RT- PCR data depend on the use of external calibrators. In this investigation, a dual target plasmid designated pRSETMON-02 comprising of MON 810 maize event specific and endogenous zein gene sequences in 1:1 ratio in tandem was constructed and validated. Commutability of plasmid DNA (pDNA) and genomic DNA (gDNA) calibrators for the quantification of MON 810 maize was assessed by employing a TaqMan RT-PCR targeting the P-35S and zein gene. Higher PCR efficiencies, good linearity and lower relative standard deviation (RSD) values were associated with pRSETMON-02 as opposed to gDNA calibrants. pDNA cali- brants exhibited better performance characteristic in terms of closeness to the expected value of unknown samples than their genomic counterparts. Short term stability study of the pRSETMON-02 plasmid stored at different temperatures showed that pDNA is stable for 45 days at 20, and 4 °C. The results demonstrated that the developed dual target plasmid pRSETMON-02 is fit for the intended use of quantifying MON 810 maize and is a better alternative to conventional seed powder calibrants. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The development and commercialization of genetically modified (GM) crops around the world has increased in an unprecedented way. Total area of cultivated land reached 160 million hectares in 2011, making GM crops the fastest adopted crop technology (James, 2011). Yet, concerns regarding their safety, environmental risks, and ethical issues among consumers still exist and need to be ad- dressed (Yang et al., 2007). Labeling regulations for genetically modified organisms (GMOs) and their products in several countries allows consumers to make informed choices. The implementation of labeling regulations necessitates the development of robust GMO quantitation methods. DNA based polymerase chain reaction (PCR) is the mainstay of GMO detection. Real time PCR (RT-PCR) is the most precise and straightforward technique for the quantitative analysis of GMOs (Gu, Wu, Li, Li, & Yang, 2009; Mattarucchi, Weighardt, Barbati, Querci, & Van den Eede, 2005). The use of external calibrators is the core for precision, sensitivity, and repro- ducibility in GMO detection and quantitative measurements (Taverniers, Van Bockstaele, & de Loose, 2004). DNA extracted from dried powder certified reference materials (CRMs) are most often used as external calibrators (Mattarucchi et al., 2005). These CRMs are developed from isogenic non-GM and GM seed powders and certified by their mass fraction. Only a limited number of GM events with limited working ranges (0–5%) are available (Lievens et al., 2010). Linearized plasmid DNA as calibrants containing either GMO specific or species specific DNA sequences are the best alter- natives to conventional CRM seed powders for such quantification (Charels et al., 2007; Folloni et al., 2010). The advantages of plasmid calibrants are that they are relatively easy to construct, stable for longer periods when frozen and can be produced, upscaled and purified in large quantities in a broader working range. The devel- opment and applicability of plasmid calibrants in quantifying dif- ferent GM events has gained momentum ever since an European Union (EU) regulation required GMO quantification to be expressed as the percentage of GM DNA copy numbers in relation to taxon specific DNA copy numbers, calculated in terms of haploid genomes (EC recommendation 2004/787). Applicability of single (Taverniers, Windels, Van Bockstaele, & de Loose, 2001), dual (Guan et al., 2011; Meng, Liu, Wang, Zhang, & Yang, 2012; Wang, Li, Yang, Shen, & Zhang, 2009) and multiple target plasmid calibrants (Debode, Marien, Janssen, & Berben, 2010; Kuribara et al., 2002; Shindo et al., 2002; Yang et al., 2007) for GMO quantification have been documented. Caprioara-Buda et al. (2012) in an inter-laboratory comparison opined that the most suitable metrological approach for GM quan- tification was to use a reference system based on plasmid DNA (pDNA) as the primary calibrant for DNA copy number. The impact of analytical quality criteria and data evaluation on the quantifica- tion of GMOs has been recently demonstrated by Meyer et al. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.02.067 ⇑ Corresponding author. Tel.: +91 821 2515331; fax: +91 821 2517233. E-mail address: lrg@cftri.res.in (L.R. Gowda). Food Chemistry 140 (2013) 382–389 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem