A novel method with improved sensitivity for the detection of peanuts based upon single-tube nested real-time polymerase chain reaction

A novel analytical method for the detection of peanuts was developed. The method is based upon single-tube nested real-time polymerase chain reaction, which uses two pairs of primers in one reaction tube, with different annealing temperatures to control the first and second rounds of PCR, and with real-time fluorogenic probe-based monitoring of the second round of PCR. The gene Ara h 3 was used as a marker specific for peanut. The developed method had an improved sensitivity compared with the initial real-time PCR, the detection limit being 0.375 pg DNA isolated from leaves. An improvement by an order of magnitude was determined also with DNA isolated from raw and roasted peanuts. The method represents a contribution to the efforts for the effective detection and quantification of peanut allergen traces in food products.