A Screening Method for the Detection of the 35S Promoter and the Nopaline Synthase Terminator in Genetically Modified Organisms in a Real-Time Multiplex Polymerase Chain Reaction Using High-Resolution Melting-Curve Analysis

Abstract

To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen ® as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

Evaluation of High-Resolution Melting for Gene Mapping in Rice

“…Furthermore, the sensitivity of HRM is very high; even a single base-pair difference can be detected (Reed and Wittwer 2004). Because of these benefits, HRM has already been used in many investigations including the detection of SNP mutations (Margraf et al 2006;Wu et al 2008), RNA editing (Chateigner-Boutin and Small 2007), identification of transgenic plants (Akiyama et al 2009), varietal identification (Mackay et al 2008), and food traceability (Ganopoulos et al 2010;Jaakola et al 2010). HRM analysis has also been successfully used for genetic mapping in white lupin, barley, apple, and almond (Chagné et al 2008;Croxford et al 2008;Lehmensiek et al 2008;Wu et al 2009).…”

A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

“…The advantages of HRM include the high efficiency and labor‐saving benefits. Since invented in 2003, this technology has been adopted in clinical chemistry, human pathology and plant sciences (White et al 2007; Croxford et al 2008; Wu et al 2008; Akiyama et al 2009; Dagar et al 2009; Hofinger et al 2009; Sun et al 2010; Ujino‐Ihara et al 2010). Several HRM platforms are available from various manufacturers.…”

“…However, the high cost of these dyes often limits the application of this technology, in particular when dealing with a large number of samples. EvaGreen (Biotium Inc., Hayward, CA, USA) is a newly developed and low‐cost dye, and has been used in quantitative real‐time polymerase chain reaction (PCR), post‐PCR DNA melt curve analysis and several other applications (Ihrig et al 2006; Sang and Ren 2006; Wang et al 2006; White et al 2007; Akiyama et al 2009; Dagar et al 2009; Sun et al 2010; Ujino‐Ihara et al 2010). It is also a so‐called saturated dye that intercalates every single nucleotide of the double‐stranded DNA.…”

A Particular Case of Novel Food

“…Numerous screening methods are based on the amplification and detection of DNA sequences found in as many different GMOs as possible [42][43][44][45][46] . The two sequences more frequently used for this purpose are the promoter, P-35S, from cauliflower mosaic virus (CaMV) and the nopaline synthase gene terminator, T-nos, from Agrobacterium tumefaciens.…”