Real-Time PCR For The Identification Of Duck Species In Foods

Real-time PCR authentication of meat species in food is justified by creating fair market environment for producers by discouraging fraud.Food authentication and quality control requires sensitive species-specific identification of DNA from unknown sources.In the last decade,methodologies for species identification in meat products are mainly based on the DNA sequences.The precision of analysis is to genus or subgenus level.A Taqman real-time PCR and a single-tube nested real-time PCR were used to be able to identify duck ingredient in food products.And a multiplex real-time PCR was developed to identify duck,pig and chicken DNA in blood curds.The contents and results were detailed as follows:1.Real-time PCR with internal amplification control for detection of duck meat in foodsFluorescent real-time PCR system for assay duck meat was constructed on the basis of a pair of primers and the TaqMan probe,specific for the duck,designed according to the sequence of duck nuclear gene(IL-2).We also designed and constructed internal amplification control(I AC)and TaqMan probe for I AC.Results showed that the specificity of TaqMan real-time PCR is reliable and it could detect the presence of duck meat even when the concentration of DNA was reduced to 0.05ng,the sensitivity of this method was 0.1%.We evaluated the effect of processing on the performance of the technique,autoclaving reduced the amplificability of duck DNA,however,boiling and high pressure processing had no effect.2.Single-tube nested real-time PCR for detection of duck meat in foodsIn order to improve the sensitivity of real-time PCR,especially for processed meat species identification,we developed single-tube nested real-time PCR systems for specific detection of duck meat and assessed the effect of processing on the performance of the techniques.The novel method enabled increasing the sensitivity.It could detect the presence of duck meat even when the concentration of DNA was reduced to 0.5pg;the sensitivity of this method was 0.01%.Autoclaving reduced the amplificability of duck DNA,however,boiling and high pressure processing had no affect.It can be suggested that the single-tube nested real-time PCR method described might be a rapid and sensitive method for identification of duck meat in raw and processed meat products.3.Multiplex real-time PCR for the identification of DNA from duck,pig and chicken in blood curdsAn accurate and reliable multiplex TaqMan real-time PCR method based on the specific sites of nuclear DNA has been developed to identify duck,pig and chicken DNA in blood curds.Total DNA in blood curd samples was extracted using three methods:Blood DNA Kit,Blood/Cell/tissue Genomic DNA Kit and phenol/chloroform extraction method.The concentration and purity of DNA was compared regarding DNA yields and purity.The phenol/chloroform extraction method and Blood/Cell/tissue Genomic DNA Kit gave improved extraction efficiencies compared with the Blood DNA Kit.The limit of detection of the assay was 0.15 ng for each target species.Analysis of experimental mixtures demonstrated that the sensitivity of the assay was 1%for each species analyzed.This system proved its accuracy and applicability for the examination of different types of animal blood in blood curd products of the type presently on the market.