Detection of Genetically Modified Canola Using Multiplex PCR Coupled with Oligonucleotide Microarray Hybridization

Abstract

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

A Particular Case of Novel Food

“…However, the application of multiplex PCR is limited by several constraints. First, multiplex PCR-based methods are more susceptible to nonspecific product amplification or cross-amplification reactions than conventional PCR since several primer pairs are added to the reaction mixture and consequently, the risk of having false positive signals might be increased [51][52][53] . Second, small differences in amplification efficiencies for the different primer pairs results in different amplification rates for the different targets.…”

Testing Methods for Agriculture and Food Safety

“…Technologies involving amplification of gene fragments by the polymerase chain reaction (PCR) have the greatest potential for detecting transgenic plants and foodstuffs derived from them (Ahmed, 2002;Schmidt et al, 2008). PCR is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism.…”

Assessment of Genetically Modified Organisms (GMO) in Meat Products by PCR

“…The detection was based on streptavidin -conjugated gold nanoparticles, and the limit of detection was below 0.3%. Schmidt et al (2008) have developed a rapid multiplex PCR -microarray method for screening GM canola. It contains probes that are construct -specifi c to the 12 approved GM canola lines in Canada, regulatory elements (CaMV 35S promoter, T -nos , and npt II), a canolaspecifi c endogenous gene, and endogenous genes from heterologous crops.…”