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Real time analysis of PCR - based DNA methods for the limit of detection,sensitivity and specificity.
Project Code: Q01033
Popping, B ;
Central Science Laboratory
The polymerase chain reaction (PCR) has become a widely used tool in many laboratories. However, there are issues that need to be addressed when using DNA methods for food authentication. Processing materials may have a degrading effect on DNA, decreasing the efficacy of some methods. Another factor is the effect of the food matrix on DNA extraction and subsequent PCR reactions, for example, the presence of PCR inhibitors or enhancers from plant material.
This project aims to assess the effect of processing and the food matrix on extraction of DNA, and PCR specificity, efficiency and accuracy. Also, to develop real-time PCR methods that will allow species-specific identification in several matrices of raw and processed foods with determined detection limits. It will focus on identification of meat and fish species.
· Use database programmes to align the turkey and pork mitochondrial cytochrome b genes and design and obtain universal primers. These will be used to amplify a region from DNA extracted from samples of raw turkey, chicken, beef and pork.
· Sequence the amplicons to determine the exact DNA sequence for the mitochondrial cytochrome b gene derived from turkey, chicken, beef and pork meats available in the UK.
· Align turkey, chicken, beef, and pork DNA sequences and design primers and probes.
· Optimise PCR (and extraction conditions) for all primers on turkey, chicken, beef and pork.
· Comparison of two real-time PCR systems used (Amplifluor and Taqman).
· DNA analysis of canned samples.
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