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Eurofins Develops Hexaplex Real-Time PCR Assay to Detect GMOs in Agriproducts

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By Ben Butkus

Scientists from Eurofins' GeneScan business have developed and validated a multiplex real-time PCR assay to screen for the presence of genetically modified organisms in food, feed, and seed.

The assay is the first example of a hexaplex real-time PCR assay using Applied Biosystems (part of Life Technologies) TaqMan probes, and is expected to reduce the time and cost associated with current GMO screening methods without compromising sensitivity, according to one of the scientists.

Eurofins GeneScan has been offering the assay as part of its testing service laboratory in Germany for the past few months, and may eventually commercialize an assay kit, the researcher said.

The assay, which is described in a paper published in the Jan. 26 online edition of Analytical and Bioanalytical Chemistry, was able to detect more than 100 approved GMOs, including all currently commercialized GM soybean and maize products, Christoph Bahrdt, a Eurofins GeneScan researcher and corresponding author on the paper, told PCR Insider in an email last week.

"The increasing complexity of genetic modifications in [GMOs] approved for commercial use and for field trials demands new time- and cost-efficient detection methods," Bahrdt said. "To fulfill legal requirements for placing food and feed products on the market, new routine testing assays have to combine broad screening methods and specific identification of GMOs lacking common screening elements."

Several multiplex assays for GMO detection using conventional PCR have been described in the literature and have met with success; however, real-time PCR offers advantages such as a higher level of automation and the integration of PCR and detection in a closed system, thereby reducing contamination, according to the researchers.

However, multiplexing real-time PCR assays presents its own set of challenges, in particular fluorescence dye crosstalk. "In a multiplex assay, the optimum combination of reporter dyes, which can vary from instrument to instrument, is of particular importance," the researchers wrote in their paper. As such, "in real-time PCR, the number of targets for multiplexing is mainly restricted to the real-time PCR platform constraints."

"To our knowledge, the highest multiplex level of a published real-time PCR assay for GMO detection based on TaqMan technology is quadruplex," the researchers added.

To overcome these limitations, the Eurofins GeneScan scientists chose a combination of fluorophores for probe labeling and filter sets for a Stratagene (now part of Agilent) Mx3005P qPCR system that would result in minimal overlap in emission spectra.

This allowed them to create an assay that could detect five GMO gene elements: cauliflower mosaic virus 35S promoter; Agrobacterium tumefaciens NOS terminator; figwort mosaic virus 34S promoter; and two construct-specific sequences present in novel GM soybean and maize that lack common screening elements. The sixth detection system was for an internal positive control that was used to indicate the presence or absence of PCR-inhibiting substances.

The PCR platform used by the researchers only has five detection channels, so the researchers used one detection channel twice. Still, there was no significant crosstalk between detection channels, according to the scientists.

Overall, the scientists demonstrated a limit of detection of no more than 10 target copies and a sensitivity of no more than 10 target copies of each GMO detection system in "highly asymmetrical target situations in the presence of 1,000 copies of all other GMO targets of each detection channel," they wrote.

What's more, Bahrdt told PCR Insider that the two modification-specific primer sets used in the process provided further information about the presence of two GMOs so far not detectable by widely used screening approaches.

Bahrdt added that the hexaplex assay "meets the current demands in routine testing," which include time- and cost-efficiency; the ability to detect GMOs in a wide range of processed and unprocessed food, feed, and seed samples; high sensitivity; low risk of contamination; and efficient processing.

Eurofins Scientific is an international group of laboratories providing testing and support services to the pharmaceutical, food, environmental, and consumer products industries.

It acquired GeneScan in 2003 in order to provide analytical tools to the agrifood industry to detect GMOs.

Bahrdt said that "the hexaplex screening assay, as published, [has been] successfully running in our routine testing service laboratory for months. He added that Eurofins GeneScan "provides a broad portfolio of kits for sale and we may introduce the hexaplex screening assay as kit in our portfolio for commercialization."

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