Q01106: Transfer of the real-time PCR method to detect and quantify common wheat adulteration of pasta to the Agilent 2100 BioAnalyser capillary electrophoresis system.
Monday 11 August 2008
Background
In order for pasta to have the correct eating characteristics (i.e. texture), it should be made from durum wheat (Triticum durum). Durum wheat normally attracts a higher price, as growing conditions are more restrictive than common wheat (Triticum aestivum). International Standard ISO 11051:1994(E) allows for 3% contamination of durum wheat with common wheat. Products labelled as durum wheat pasta which contain more than 3% common wheat will mislead consumers and thus will contravene the Food Labelling Regulation 1996.
A real-time polymerase chain reaction (RT-PCR) method for detecting and quantifying adulteration of durum wheat with common wheat was developed and validated for the Agency. RT-PCR is a powerful technique which amplifies specific DNA fragments in proportion to the amount present and allows for accurate detection and quantification of the target species. However, most public analyst (PA) labs do not have RT-PCR, and this project is one of a number of projects commissioned by the Agency to transfer existing DNA based methods to a simple yet robust lab-on-a-chip (capillary electrophoresis) system which most UK PA labs have access to.
Research Approach
The genetics of common wheat and durum wheat differ as common wheat has three sets of genomes (hexaploid) whilst durum wheat has only two sets of genomes (tetraploid). The additional genome in common wheat is known as the D-genome. The RT-PCR method amplifies and measures common wheat adulteration in durum wheat pasta by comparing the number of copies of two specific DNA sequences, one which is unique to common wheat from the D-genome, and the other which is common to all wheat genomes, known as a normalisation sequence. The aim of this project was to transfer the RT-PCR method to the lab-on-a-chip system.
Results and findings
In order to detect adulteration, the lab on a chip system requires separation of the PCR products from the target and normalising sequences. To achieve this, the normalising sequence had to be extended slightly in length, enough to permit separation without disturbing the PCR kinetics. A semi-quantitative method has been developed which detects if more than 3% common wheat has been used in durum wheat pasta. A standard operating procedure has been written detailing this procedure.