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1、生物工程学报ChinJBiotech2009,January25;25(1):139-146journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn©2009InstituteofMicrobiology,CAS&CSM,Allrightsreserved生物技术与方法多重实时荧光PCR检测牛、山羊和绵羊源性成分曾少灵,秦智锋,阮周曦,花群义,卢体康,吕建强,陈书琨,曹琛福,张彩虹,孙洁,陈兵,吴绍精深圳出入境检验检疫局,深圳5180
2、45摘要:根据牛、山羊和绵羊线粒体细胞色素b基因序列,设计特异性引物和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,建立能同时鉴别牛、山羊和绵羊源性成分的多重实时荧光PCR方法。采用本文方法与国标GB/T20190-2006方法分别对17种不同源性动物DNA和200份不同来源样品DNA进行牛羊源性成分检测,数据显示两者检测结果符合率达100%,特异性相当。与国标方法相比,本试验方法不需电泳、酶切和测序,即可在一个PCR反应中同时鉴别检测牛、山羊和绵羊3种源性成分,检测效率提高近3倍;灵敏
3、度更高,比国标方法灵敏10倍;适用性更广,除了饲料,还适用于肉品、奶品、生皮和动物油脂等动物产品的牛羊源性成分检测。关键词:细胞色素b基因,多重实时荧光PCR,牛、山羊和绵羊源性成分Multiplexfluorescentreal-timePCRdetectionofbovine,goatandsheepderivedmaterialsinanimalproductsShaolingZeng,ZhifengQin,ZhouxiRuan,QunyiHua,TikangLu,JianqiangLü,ShukunChen,C
4、henfuCao,CaihongZhang,JieSun,BingChen,andShaojingWuShenzhenEntry-exitInspection&QuarantineBureau,Shenzhen518045,ChinaAbstract:WedesignedthespecificprimersandTaqManprobestargetingcytochromebgenesofmitochondrialDNAfrombovine,goatandsheep.Weuseddifferentfluorescent
5、stolabeltheprobes.Afteroptimizationofreactionconditions,wesetupamultiplexfluorescentreal-timePCRmethodtodetectbovine,goatandsheepderivedmaterials,simultaneously.Wefinishedthedetectiontestsof17kindsofanimalDNAand200DNAsamplesfromdifferentsourceswiththedevelopedme
6、thodandtheNationalStandardGB/T20190Y-2006routinePCRmethod.Thecoincidencerateofthesetwomethodswas100%.Withoutelectrophoresisorrestrictiondigestion,thedevelopedmethodcouldreducethetesttimetoonethirdasroutinePCRandidentifythreekindsofanimalderivedmaterialsincluding
7、bovine,goatandsheepinonereaction.Thedevelopedmethodwasapproximately10timesmoresensitivethanroutinePCR,andwasapplicabletoidentificationsofbovine,goatandsheepderivedmaterialsinfeedstuff,meat,milk,peltandgrease,etc.Thestudyshowedthatthedevelopedreal-timePCRmethodis
8、arapid,sensitiveandefficaciousdetectionassayforbovine,goatandsheepderivedmaterialsinanimalproducts.Keywords:cytochromebgene,multiplexfluorescentreal-timePCR,bovine,go
