Alternative reference material for quantitative PCR

OBJECTIVES:

Certified reference material for quantitative GMO testing is only available in the form of mixtures of ground GM and non-GM substances at set ratios. Ideally, such material should be in the form of DNA. For this purpose, this project assesses the suitability of using purified genomic DNA and plasmidic DNA as quantitative PCR reference material.


APPROACH:

Electron micrograph: Two circular strands of DNA known as plasmids
Electron micrograph: Two circular strands of DNA known as plasmids
This task compares the calibration behaviour of DNA extracted from a plant versus DNA synthetically produced in the lab. DNA from the plant is considered genomic DNA, because all of the DNA in the plant's genome is extracted together. DNA made in the lab, however, would only actually need to possess the sequences targeted by the PCR test. The easiest way to produce the target genes in a lab is to culture bacteria that harbour small, circular pieces of DNA called plasmids. Therefore, this project is comparing the effectiveness of extracted, genomic DNA with synthetic, plasmidic DNA. Ideally, both types of DNA will be suitable as reference material.

In order to get an unbiased comparison, samples of grain material with unknown GM content are analysed by five different laboratories using each of the types of DNA as reference material. Measurements are spaced out over an extended time period, to simultaneously test the reference material's stability.

Once the PCR tests are complete, the performance of genomic and plasmidic reference material is compared. It is hoped that both types of reference material will behave similarly and provide similar results. Ideally, both will be able to be used in any procedure without affecting any other steps or the end results.


SHORT RESULTS:

A detailed measurement protocol has been developed in co-operation with the five participating laboratories. A first set of measurements was made in April 2006 and is currently being evaluated. The final results of the evaluation of the calibration behaviour are expected by October 2007. Comprehensive results will be communicated via scientific publications.

More information:

Report on the benefits and limits of pJanus and MTPs as quantitative calibrants
This report reviews the cloning and application of plasmid DNA markers usable as positive controls. Provided the suitability of plasmids as calibrants in GMO analysis can be demonstrated, plasmids would represent an ideal solution to overcome the current lack of independent calibrants.

Reference materials and reference PCR assays for GMO quantification



PARTICIPANTS:

NAME / ORGANISATIONCONTACT INFORMATION
Isabel Taverniers
Institute for Agricultural and Fisheries Research (ILVO), Belgium
Email: info@coextra.eu
Amaya Leunda Casi
Institut Scientifique de Santé Publique (ISP), Belgium
Kristina Gruden
National Institute for Biology (NIB), Slovenia
Andreas Wurz
Genescan, Germany
Stefanie Trapmann
Institute for Reference Materials and Measurements (IRMM, part of the Joint Research Center), Belgium