Study On Detection The Avian And Fish-derived Constituents In Feedstuffs By Real-time Pcr And Pcr-dhplc

It is important issues for feed safety and environmental bio-security which affect sustainable development and economic development of China. The major issues are food-origin disease and environmental hazard caused by feeds contamination. To guarantee food security and enhance international competition of feed and food products of China, it is necessary to research and establish efficient, durable, accurate, high-throughput detection technology for animal-derived constituents.After the agarose gel electrophoresis and concentration, purity testing of genome DNAs extracted by kit and cetyltrimethylammonium bromide(CTAB) methods, the CTAB method were choosed for animal genomic DNA extraction.According to the conserved mitochondrial DNA sequences, the specific primers and probes for real-time PCR and the specific primers for PCR-DHPLC are designed. After analyzing the sequences by blast and optimizing the detection conditions of PCR and the analysis conditions of PCR-DHPLC, new detection systems for identifying avian and fish materials in feedstuffs using real-time PCR and PCR-DHPLC formats are established. The results show that:(1) The mtDNAs of chicken, duck, goose, partridge and fish are amplified successfully by the real-time PCR, but no amplification from other animals; Chicken and goose materials can be detected by the PCR-DHPLC and no amplification from other animal-components.(2) The detection limits of real-time PCR for chicken, duck, goose, partridge and fish are 0.1%,1%,0.05%,0.1%,0.1%; The detection limits of PCR-DHPLC detecting system for chicken, goose are 0.1% and 0.01%, which reached the detection limit requirements of 0.01% from the Codex Alimentarius Commissionand and the International Veterinary Bureau except the detection limit of duck using real-time PCR.(3) Amplification curve of real-time PCR from three parallel samples overlapped, and the coefficient of variation (CV)of Ct values were less than 2%. The detection work can be finished just in 1.5 hour. The same eluting peaks of PCR-DHPLC from 5 repetition-tests were gotten.(4)2 feedstuff samples are detected by the real-time PCR and PCR-DHPLC detecting system, and detecting result shows that 8 samples contain chicken-derived materials,3 samples contain duck-derived materials,2 samples contain goose-derived materials, but no positive samples of partridge and fish-derived materials, which match of 100% with the investigation results.Overall, this real-time PCR and PCR-DHPLC detection methods are sensitive with good specificity and repeatability, both can be used for animal-original feed and food testing.