Multiplex PCR

OBJECTIVES:

Conventional PCR tests for GMOs only test for one GMO at a time. With more and more GMOs being developed, tests for will only be getting more expensive. The need for multiplex GMO tests, which would detect many GMOs at the same time, is therefore acute. Co-Extra researchers are now working on ways to detect the presence of many GMOs at once. Although multiplex detection is within reach, simultaneously determining their quantities remains a great technical challenge.


APPROACH:

The first concern with developing multiplex PCR methods is identifying the gene sequences that the test will target. Research are compiling an ongoing list of genetic sequences that are characteristic for certain crops and GMOs.

The genetic sequences that can be targeted include the sequences of specific transferred genes or sequences for the commonly used genetic building blocks that often accompany transgenes. Other useful gene sequences are those that code for genes specific to a certain crop. That way, a test can determine if a food contains unexpected traces of crops like soybeans, barley, or rice.

Flourescent probes are used in quantitative PCR to track how fast a gene of interest is being copied. To simultaneously measure the amounts of different certain genes in a single sample, Co-Extra researchers are tagging target genes with different coloured fluorescent probes. That way, signals coming from different target genes can be isolated to find out exactly which of the GMOs have been found.

To start, researchers are taking genes specific to barley or wheat and are measuring their quantities in duplex - which means measuring the quantity of two genes simultaneously.


SHORT RESULTS:

New multiplex approaches have recently been designed for simultaneous detection of very high numbers of target sequences: these can be considered as high-grade multiplex approaches. Some of them include a first ligation step that is dependent upon hybridization of two oligonucleotide sequences to the target, subsequent amplification (with universal primers) and detection by hybridization on array support. Examples are a SNPlex method (for single nucleotide polymorphisms detection) designed to identify GMO targets; and a system based on padlock probes (circularizable probes). In a very different approach, a whole genome amplification (WGA) technique can allow producing large amounts of genomic DNA of the sample that are then hybridized to special probes in microtiter plates or microarrays to detect GMO targets (e.g. high density tilling microarray).


More information:

New multiplexing tools for reliable analysis of GMOs

Detecting unauthorised and unknown GMOs

Validation of novel methods and technologies 

DualChip GMO - a new tool to facilitate the screening for GMO in the food and feed chain

Public Deliverables of the Co-Extra project



PARTICIPANTS:

NAME / ORGANISATIONCONTACT INFORMATION
Arne Holst-Jensen
National Veterinary Institute (NVI), Norway
Email: info@coextra.eu
Knut Rudi
Matforsk AS, Norway
Isabel Taverniers
Institute for Agricultural and Fisheries Research (ILVO), Belgium
Doerte Wulf
GeneScan Analytics GmbH, Germany
Christophe van Huffel
Eppendorf Array Technology (EAT), Belgium
Maria Pla
Consejo Superior de Investigaciones Científicas (CSIC-IRTA), Spain
Marleen Harink
RILKIT Institute of Food Safety, The Netherlands
Dany Morisset
National Institute of Biology (NIB), Slovenia
Institut National de Recherche Agronomique (INRA), France