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The development of PCR based method for the identification of peanut in commercial products
Project Code: T07024
The adventitious adulteration of commercial food products with allergen bearing foods is of great concern to both food producers and allergy sufferers. A method of identification is required therefore, which is simple, sensitive and specific for the allergenic food.
The objective of this 1-year study was to develop a sensitive and robust assay for the identification of peanuts in commercial products.
The assay was based upon the amplification of peanut specific sequences with PCR primers followed by visualisation using an ELISA format designed to detect double stranded DNA.
A range of commercial DNA extraction kits was assessed for their ability to efficiently extract PCR quality DNA from peanuts. Taqman technology for the detection of peanut DNA was used. Three Taqman primer and probe sets, specific for peanut sequences in the genes Arah2, NGLB1 and CHI, were designed.
The Taqman primer and probe sets were optimised according to the protocol suggested by Perkin Elmer. The most sensitive (lowest Ct) primer and probe set with the greatest dynamic range was specific for the Ara h2 gene. This primer and probe set was used for the remainder of the project.
The specificity of the Ara h2 primer and probe set was determined using 33 food items including peas, beans, tree nuts and mammalian and avian tissues as template DNA. This primer and probe set did not cross react with any of the food items tested.The limit of detection (LOD), assessed using dilutions of peanut DNA, was determined to be equivalent to a 1:100,000 dilution of peanut DNA. The accuracy of the assay was determined using commercial samples, which had either come into contact with peanut or were spiked with 10% (w/w) of peanut. It was found that both the trace and spiked samples were determined as positive using the assay.
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