饲料用鱼类、哺乳类及禽类源性成分三重TaqMan-MGB荧光PCR检测方法的建立及应用  

Establishment and application of a Triple TaqMan-MGB fluorescent PCR detection method for fish,mammal and avian derived materials in feeds

作　　者：吕继洲 袁向芬[2] 林祥梅[2] 林玉[1,2] 邓俊花 王慧煜 孔玉方[1,2] 吴绍强 LYU Jizhou;YUAN Xiangfen;LIN Xiangmei;LIN Yu;DENG Junhua;WANG Huiyu;KONG Yufang;WU Shaoqiang(CAIQ Center for Biosafety in Sanya,Sanya Hainan,572024,China;Chinese Academy of Inspection and Quarantine)

机构地区：[1]三亚中国检科院生物安全中心,海南三亚572025 [2]中国检验检疫科学研究院

出　　处：《质量安全与检验检测》2023年第3期16-24,共9页QUALITY SAFETY INSPECTION AND TESTING

基　　金：“十四五”国家重点研发计划项目(2022YFF0608201)。

摘　　要：为建立饲料用鱼类、哺乳类及禽类源性成分的广谱、快速检测方法,收集Genbank中鳕鱼、鲈鱼、沙丁鱼等鱼类,猪、牛、羊等哺乳类以及鸡、鸭、鹅等禽类的18S rRNA基因序列,分别设计特异性扩增引物和MGB-TaqMan荧光探针,通过优化反应体系和条件,每种引物探针体系均可以特异性检测目标物种类型而与其他物种无交叉反应。3套引物探针体系组合后可同时检测鱼类、哺乳类及禽类源性成分,三重荧光PCR方法的最低检测限可低至0.002 ng或0.02 ng基因组/反应,变异系数均小于2%。利用建立的三重荧光PCR方法检测35份进境及国产饲料样本,从2份进口饲料及1份国产饲料中检出非标定动物源性成分,结果与行业标准方法的检测结果一致,符合率100%。结果表明,本研究建立的三重荧光PCR方法具有特异性、敏感性高、重复性好等优点,为包括饲料在内的动物产制品源性成分定性分析提供了新的技术分析手段。In order to establish a broad-spectrum and rapid detection method for the components of fish,mammals and poultry,18S rRNA gene sequences of fish(cod,sea bass and sardines et al.),mammals(pig,cattle and sheep et al.),and poultry(chicken,duck and goose et al.)were collected from GenBank.Three sets of specific amplification primers and MGB-TaqMan fluorescent probes were designed respectively based on the above-aligned sequences.By optimizing the reaction system and conditions,each set of primers/probe could specifically detect the targeting animal species without cross-reaction with other species.The Triple fluorescence PCR method was established based on the three sets of primers/probe.The established PCR method could simultaneously detect the fish,mammal and avian derived materials and the minimum detection limits could be as low as 0.002 ng/reaction or 0.02 ng/reaction.The variation coefficient was less than 2%.35 imported and domestic feed samples were detected by the established method and it was found that non-labeled animal-derived materials were detected in 2 imported feed samples and 1 domestic feed sample.The detection results were consistent with the detection methods described in the standards,with a coincidence rate of 100%.The results showed that the triple RT-qPCR method established had the advantages of specificity,high sensitivity and good reproducibility,and provided a new technical analysis method for the qualitative analysis of animal products such as feeds.

关 键 词：哺乳动物源性成分 鱼类源性成分 禽类源性成分 荧光PCR 

分 类 号：S816.17[农业科学—饲料科学]