Development And Application Of A PCR Approach For Detection Of Animal Derived Materials In Feedstuffs

In order to improve the existing method for detection of animal derived materials in feedstuff, this paper described the development of species specific PCR assay to detect bovine, sheep, porcine, chicken and fish content in feedstuffs. Meanwhile, through the investigation and random sampling, a preliminary survey of the use of animal feed in dairy was got. This paper includes three parts.First, an investigation was carried out in He Bei provinece, Hei Longjiang province and Baotou city. 201 farmholders were involved in the survey. The results showed that 9 farmholders added meat and bone meal in feedstuffs, accounted for 4.48% of total farmholders.Second, a PCR aproach was developed to detect animal derived materials in ruminant feedstuffs.DNeasy Tissue Kit of Qiagen was applied to extract nucleic acid. General primer designed according to 16S rRNA gene sequence of bovine, sheep, porcine, chicken, fish and horse mtDNA was used for primary detection of animal derived materials in feedstuff. Bovine and sheep primers which have been reported were used in the research. Primers Pig9044/9066F/Pig9294/9314R and Chicken9129/9151F/Chicken9305/9327R were designed according to ATPase8 special region on mtDNA of pig and chicken.Primers NC_Fish2480/2501F/NC_Fish2565/2586R were designed according to mitochondria 16S rRNA sequence. Specificity detection results showed well specificity of the bovine,sheep,porcine,chicken,fish primers, which were used for amplification of a 271bp, 275bp, 271bp, 199bp and 107bp fragment respectively. Sensitivity results showed that the detection limitation was 0.1% .The positive samples detected with bovine-derived ingredients were further analyzed with chloroform and 0.5% sodium hypochlorite to identify whether the bovine-derived ingredients were from milk or meat-and-bone meal.Third, 364 samples of ruminant feedstuff and animal feed were totally collected from Beijing, Tianjing, Hebei provinece and so on. And then these samples were detected by the PCR assay in this study. Animal-derived materials were detected in 66 samples, accounted for 18.13% of the total samples. Bovine and sheep-derived materials weren't detected in ruminant feedstuffs, while porcine and fish-derived materials were found. Bovine,sheep,porcine,chicken and fish derived materials were detected in animal feedstuffs.