The exchange of knowledge in the framework of ENTRANSFOOD between the RTD projects QPCRGMOFOOD and GMOBILITY:
Extraction and detection of nucleotide sequences in various matrices
Jos van der Vossen, Coordinator GMOBILITY project
Aim of the exchange of information was to allow both RTD projects to take advantage of the knowledge built in these projects under the umbrella of ENTRANSFOOD. In both projects it is of importance to have efficient DNA extraction methods available. For the QPCRGMOFOOD project it is essential to detect non-equivalent DNA sequences in various food matrices in order to identify a food product containing GMO material for labeling reasons. For the GMOBILITY project detection of DNA is essential for the determination of the persistence of DNA in food-, feed-, and gastrointestinal matrices as well as the detection of bacteria that acquired DNA via horizontal gene transfer. Both projects could implement the gained knowledge on DNA extraction and purification methods for their research aims.
Place of venue was the Institute für Mikrobiologie und Genetik, University of Vienna, Austria at the 26th of March 2001. The meeting started at 10.00 and closed at 13.00. After introduction of participants of the GMOBILITY meeting as well as introduction of Dr. Carlo Bulkmans as a representative of the WG1 of the QPCRGMOFOOD project both a presentation of Dr. Carlo Bulkmans and experiences of the partners of the GMOBILITY project were presented. Based on the presentations various strategies for the isolation of DNA were discussed.
New ideas were the result of the presentations dedicated to the sample pretreatment work of both projects. Aspects such as problems encountered, practical solutions, and strategies tested that were dedicated to specific matrices were discussed. The presentation of Carlo Bulkmans from QPCRGMOFOOD presented the experience of Workpackage 1: identification of scope and matrix limitations for a standard DNA extraction protocol. Generic methods were based on testing several possible variables and subsequent evaluation directed to samples that covered the most important matrices including fat rich, protein rich, carbohydrate rich, phospholipids and end products. Most important steps identified being essential were lysis, purification and concentration. The technical approach depends highly on the nature of the sample. Typical strategies included lysis of the matrix (fatty matrix is an exception since lipase is not effective), DNA binding by DEAE or Silica, extraction chloroform to get remove inhibitory compounds, precipitation / concentration DNA with ethanol of Silica. All strategies are dedicated to an optimal PCR detection thus balancing between quality and quantity of DNA. Available state of the art extraction procedures were identified as a good starting point. This was also recognized in the GMOBILITY project, where various state of the art methods both commercial and published methods were implemented for the detection of DNA in the various matrices encountered in the project such as food, feed, gastric and intestinal fluids, and feces. Take home lessons from the QPCRGMOFOOD project will be considered and implemented in the ongoing research where appropriate.