饲料中牛、羊、猪、鸡源性成分的PCR检测方法及其应用

 【目的】建立鉴定饲料中牛、羊、猪、鸡源性成分的PCR方法，进一步完善现有的动物源性饲料检测方法。【方法】根据牛、羊、猪、鸡、鱼以及马的mtDNA的16S rRNA基因序列选用了一对通用引物，初步检测出含有动物源性成分的样品；然后针对牛、羊、猪、鸡4种动物线粒体mtDNA中的保守序列，分别选用了4对特异性引物对检测出的样品进行PCR扩增，扩增的目的片段大小分别为271、274、149和266 bp。【结果】通过利用通用引物、优化PCR反应体系及其条件，建立了从未知样品中简便而快速的检测出是否含有动物源性成分的PCR方法；利用所选的针对不同动物的特异性引物，建立了鉴定牛、羊、猪、鸡这4种动物源性成分的方法，此方法最低检测限可达0.1%。【结论】建立的检测饲料中动物源性成分的方法操作简便，价格低廉，结果准确，可靠，可作为饲料中动物源性成分的鉴定方法之一。

Development and Application of a PCR Detection of Bovine,Sheep, Pig and Chicken Derived Materials in Feedstuff

【Objective】To improve the existing method and develop a fast, precise and reliable PCR method for detection of animal derived materials in feedstuff. 【Method】Firstly, general primer designed according to 16S rRNA gene sequence of bovis, sheep, pig, chicken, fish and horse mtDNA was used for primary detection of animal derived materials in feedstuff. Species-specific primers designed according to conserved sequence of mtDNA of bovis, sheep, pig and chicken were used for amplification of a 271bp, 274bp, 149bp and 266bp fragment respectively. Further confirmation of the detection result was then performed. 【Result】PCR method for detection of animal derived materials in unknown feedstuff was developed by using general primer, relevant PCR system and PCR condition. Also a PCR method for detection of each species (bovis, sheep, pig and chicken) was designed by using our species-specific primers. High sensitivity and specificity of our method was confirmed with a minimum detection level of 0.1%. 【Conclusion】Method for detection of animal derived materials in this research is not only cheap and easy for operation, but also precise and reliable for result. It could be one of the effective methods for detection animal derived materials in feedstuff.