HOHENHEIM UNIVERSITY

INSTITUTE OF FOOD TECHNOLOGY
General Food Technology
and Microbiology
Prof. Dr. W. P. Hammes

 

 

 

Report of experience                                                                   

Title :
Development of a real-time PCR method for the detection of PCR fragments of various length

Partner institutes :
Hohenheim University, Germany, (GMObility; QLK1-CT-1999-00527)
National Veterinary Institute, Norway (Qpcrgmofood; QLK1-CT-1999-01301)

Participant :
Torsten Bauer, Hohenheim University

Within the recent years, several real-time PCR methods for the detection and quantification of GMO contents in foods have been established and validated. Remarkably all established methods are focussed on a fragment length of approximately 100 to 200 nt. The main objective of the stay at the National Veterinary Institute (NVI) in Oslo was to develop and to optimise a real-time PCR system for long PCR fragments. This system will be used in future projects for the monitoring of the degradation of DNA during the processing of food. Several PCR systems for the detection of Roundup Ready^© soy (RRS) were tested. PCR fragments within a range of 645 to 895 nt could be amplified. The detection was performed with Taqman probes (Berdal et al. 2001) and the LightCycler (Roche). Primers and probes were targeted against the lectin gene (le1) and the genetic modification of the RRS, respectively. Best results were obtained for the amplification of a 714 nt PCR fragment on the le1. The PCR system was then optimised by investigating and adjusting the optimal reaction temperature, MgCl₂ concentration and extension time. After adjusting, detection was successful within a range of 6 orders of magnitude. Accuracy and reliability were tested by performing several runs in duplicates or triplicates and were considered as sufficient. A single copy can still be detected with this method and if 10 or more copies were subjected to PCR, the calculated coefficient of correlation was optimal.