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Nestlé R&D Subsidiary Develops Multiplex Real-Time PCR Method for HT Identification of GMOs

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Researchers from Nestec, an R&D subsidiary of Nestlé, have developed and validated a multiplex real-time PCR method to simultaneously detect 47 targets to aid in identifying genetically modified organisms.

The assay method, which the company developed primarily for internal use, theoretically enables the screening of about 95 percent of the worldwide known GMOs described in public databases, according to the scientists.

In addition, the method is fast, easy to use, and relatively inexpensive compared to existing methods for detecting GMOs, and thus could be a boon to GMO testing laboratories, Geoffrey Cottenet, a scientist at Nestec who helped develop the assay, told PCR Insider this week. As such, Nestec is making the test method available to other interested organizations.

Cottenet said that Nestec developed the assay protocol as a tool to help Nestlé comply with regulations related to GMOs.

"In the US, it's not really yet the case, but in lots of countries worldwide, there are regulations related to … the presence of GMOs," he said. "We have to comply with these regulations, and of course we have to comply with the preference of consumers related to the presence of GMOs."

Cottenet and colleagues described the protocol in a paper published this month in Analytical and Bioanalytical Chemistry.

According to the researchers, many methods have been developed to ensure authenticity and traceability of genetically modified material. These include 2D electrophoresis and isoelectric focusing, protein capillary electrophoresis, high-performance liquid chromatography, and enzyme-linked immunosorbent assays.

"However, in order to overcome limitations related to heat-treated or processed products, the use of DNA-based methods and especially PCR techniques is preferred for both raw ingredients and processed food," the researchers wrote. "Furthermore, PCR and real-time PCR are internationally recognized and recommended for GMO analyses."

Indeed, the International Organization for Standardization detailed in ISO 21569:2005 and ISO 21570:2005 methods for qualitative and quantitative detection of GMOs and GMO-derived products, focusing on PCR-based methods.

And while PCR methods have been successfully employed, the Nestec researchers were seeking a way to make the method amenable to higher-throughput screening of GMOs while retaining accuracy and assay robustness.

"The limit here was the number of targets," Cottenet said. "We had to adapt [the assay] to the 384-well plates that we usually use. That's why we switched to … multiplexed triplex systems."

More specifically, Nestec worked with Life Technologies to create custom primers and minor groove binder TaqMan probes using Life Tech's Primer Express 3.0 software. The TaqMan probes were 5'-labeled with 6-carboxyfluorescein (FAM) or VIC fluorophores and 3'-labeled with a non-fluorescent quencher.

Cottenet said that the researchers chose the minor groove binder TaqMan technology because it offers a higher degree of sensitivity and specificity than many alternative PCR chemistries.

Nestec and Life Tech then lyophilized assays columnwise on 384-well plates at a final concentration of 900-nM primers and 250-nm probes for each target. Each well contained a FAM- and VIC-labeled target — enabling detection of pertinent plant and GM markers — and a NED-labeled target — an internal positive control for the detection of potential PCR inhibition.

The researchers performed real-time PCR on an ABI Prism 7900 Sequence Detection System from Life Tech.

To enable efficient detection and identification of GM material, the group selected three different categories of targets: plant endogenous sequences to specifically detect soya, maize, rice, wheat, rapeseed, potato, cotton, and tomato; various key indicators of GM material, such as cauliflower mosaic virus 35S promoter and figwort mosaic virus 35S promoter; and construct-specific assays designed to avoid SNP occurrences and identify 28 GM events.

Regarding the triplex multiplexing combinations in each assay well, Cottenet said that the group simply found combinations that worked through trial and error, "and it works on the majority of the combinations, except for three or four combinations, and there we had the assistance of the bioinformatics team at [Life Tech] to help us identify which probe should be slightly modified or switched by several nucleotides. It worked fine on the majority of combinations."

By testing non-GM materials, different GM events, and proficiency test samples, the researchers found that their assay had a high degree of specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target.

The assays were also able to detect potential plant cross-contamination and simultaneously screen seven routine samples to obtain their global transgenic fingerprint and content, the researchers reported.

Overall, the researchers wrote, the assay is "fast, specific, sensitive, and straightforward," and is ideal for "GMO testing laboratories complying with the analytical requirements described in ISO 24276:2006," which specifies standards for nucleic acid extraction and qualitative and quantitative nucleic acid analysis for use in analyzing GMOs in foodstuffs.

Cottenet said that theoretically the assay could be developed for any real-time PCR platform with the 384-well format, but that Nestec continues to work with Life Tech for its assays.

"We will use this internally, and of course we will try and update and improve it as much as possible in coming years, because we have to adapt to the new GMOs that are being developed," he said. However, the company is leaving the assay protocol open to other entities "because it is part of our policy to try and collaborate with external parties," Cottenet said. "It could even be authorities or government laboratories."

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