中文摘要：

旨在建立一种可同时检测猪、牛、羊、鸡、鸭、鹅6种动物源性成分的六重实时荧光定量PCR方法。根据猪、牛、羊、鸡、鸭、鹅细胞核基因组保守序列,参照序列比对结果选择差异位点设计6对特异性引物和6条以不同荧光素标记的Taqman探针。通过对PCR反应体系和反应条件的优化筛选,建立能同步定量检测猪、牛、羊、鸡、鸭、鹅源性成分的六重实时荧光PCR方法。应用此方法分别对18种不同源性动物DNA和200份不同来源样品进行猪、牛、羊、鸡、鸭、鹅源性成分检测,结果表明,所建立的六重实时荧光PCR方法灵敏度高,对六种动物的最低核酸检测量分别为0.049ng、0.048ng、0.085ng、0.13ng、0.162ng、0.074ng（50μl体系）;特异性强,对狗、兔、鼠、驴、马、骆驼等其他12种动物无特异性扩增;200份样品的检测结果表明六重qPCR检测方法敏感,同一反应体系下实现一次对6种动物快速定量检测,耗时短、效率高、适用性广,可用于肉品、奶品、皮毛和饲料等动物产品猪、牛、羊、鸡、鸭、鹅源性成分单一或混合物种的鉴别诊断。

英文摘要：

Objective： To establish a highly efficient method for simutaneously detecting pig,bovine,sheep,chicken,duck and goose derived materials in animal products by use of the multiplex fluorescent real-time quantitative PCR（ qPCR）. Methods： The species-specific primers and probes for detection of beta-actin（ actb）genes of pig,bovine,chicken,duck and goose,and the prolactin receptor gene of sheep in multiplex fluorescent real-time quantitative PCR assay were designed. Total DNA as templates were extracted respectively from the six kinds of animal materials. The specificity and sensitivity of these primer pairs and probes were separately confirmed using simplex real-time qPCR analysis. Six pairs of primers and six fluorescent probes were mixed up with the above 6 kinds of animal templates. And the most effective reaction system and condition for multiplex（ hexa-plex） qPCR method were obtained experimentlly after lots of tests and optimization. The specificity,sensitivity and accuracy of this assay were evaluated. Results： The limits of detection（ LOD） of the assay were0. 049 ng,0. 048 ng,0. 085 ng,0. 13 ng,0. 162 ng,0. 074 ng DNA for pig,bovine,sheep,chicken,duck and goose,respectively（ 50μl qPCR reaction system）,and the amplification results of other animals were all negative. A total of 200 specimens tested by the multiplex qPCR method one more showed its accuracy,precision and high efficiency. Conclusions： The developed multiplex fluorescent real-time quantitative PCR method was a rapid,specific,sensitive and efficacious detection assay for pig,bovine,sheep,chicken,duck and goose derived materials in animal products,and was applicable to identifications in feed stuff,meat,milk,pelt and grease,etc.