Detection Of Animal-derived Ingredients With Universal PCR-microarray Method

The animal-derived ingredients in feedstuff and foodstuff is the main approach of spreading of Bos spongiform Encephalopath, Avian Influenza, and H1N1. Microscope analysis, near infrared spectroscopy, immunoassay analysis, and polymerase chain reaction applied in the world for the detection and identification of animal-derived ingredients in feedstuff and foodstuff. There are some disadvantages of these methods, e.g. the much experience of operators, small thoroughtput, and false positive results. The method of microarray is more rapid, accurate and economical than them. This paper aims to develop a new reliable, specific and sensitivity method for identification of animal-derived ingredients in feedstuff and foodstuff by using microarray combination with a pair of universal primers to avoid the imported animal-derived ingredients in feedstuff and foodstuff prevent China from Bos spongiform Encephalopath, Avian Influenza, and H1N1.We analyzed the sequences of vertebrate molecular marker genes, then selected the mitochondrial DNA (mtDNA) 16S rRNA gene as marker gene. In order to detect sixteen kinds of animal-derived ingredients, which including bos, goat, sheep, deer, bubalus, donkey, pig, dog, rabbit, chicken, duck, goose, quail, turkey and fish. We utilized a pair of universal primers, designed sixteen sets of species-specific microarray probes and two pairs of quality control probes. After optimization, the woking conditions were designated as follows. The concentration of spotting solution was 40μmol/L; the hybridization temperature was 52℃; and the hybridization time was 2 h. These sixteen kinds of animal-derived ingredients could be rapid and accurate detected by this approach. The detection limits were all reaches 10 pg. We established the detection platform of these sixteen kinds of animinal-derived ingredients. This universal PCR-microarray assay provides a new method for the identification of animal-derived ingredients in the import-export field. The established method was applied to detect 5 samples of sheep. The results presented positive, which were consistent with the results of PCR completely. Eight blind samples were tested using this microarray detection method, the results were also consistent with conventional PCR.Finally, a rapid, specific, sensitive and stable microarray method for general detection of animal-derived ingredients was established in this study, which can be applied to detection whether it contains animal-derived ingredients in feedstuff. And this detection method can also apply to the detection of animal products. It will play a key role in preventing the Bos spongiform, Avian Influenza, and H1N1 into China, keeping national economic safety and peple healthy and promoting the development of feedstuff and food industry.