常见淡水鱼肉制品快速鉴别方法的建立

近年来,随着肉制品掺假问题日益突出,特别是鱼肉制品中掺入廉价成分,混淆鱼肉品种,以次充好的掺假造假问题引起广泛关注。为解决这一问题,有必要针对常见的青鱼(Mylopharyngodon piceus)、草鱼(Ctenopharyngodon idellus)、鲢(Hypophthalmichthys molitrix)和鳙(Aristichthys nobilis)四大家鱼建立快速同时检测鱼肉制品的多重PCR方法。实验通过对青鱼D-LOOP基因、草鱼COI基因、鲢16SrRNA基因和鳙12SrRNA基因序列进行对比分析,针对特异序列设计4对引物,对特异基因进行单一PCR和多重PCR扩增,根据其特异性、敏感性建立并摸索L9(34)正交试验单管多重PCR最佳反应条件,包括引物浓度、Tm值、Mg2+浓度和模板量等,通过对PCR条件的优化,建立快速检测青鱼、草鱼、鲢和鳙四大家鱼鱼肉制品的单管多重PCR鉴定方法。结果表明,针对青鱼、草鱼、鲢和鳙4种鱼肉制品所设计的4对特异引物分别能扩增出298、210、306及639 bp的目的条带,具有高度特异性,反应条件优化后,4种鱼肉制品所含靶基因质粒在50 pg均可同时扩增出较清晰条带,以人工自制鱼肉制品进行了验证,结果表明所建立的方法,能够区分混合样品中4种鱼肉的构成,并对市售鱼肉制品进行检测,结果稳定。本研究所建立的方法能够简单、快速地对青鱼、草鱼、鲢和鳙4种鱼肉制品成分进行检测和监控,且特异性强、灵敏度高,可为鱼肉制品质量安全提供技术支持。

Establishment of a rapid identification method for common freshwater fish meat products

The study aims to establish a multiplex polymerase chain reaction(PCR)assay for identification of Mylopharyngodon piceus,Ctenopharyngodon idella,Hypophthalmichthys molitrix,and Aristichthys nobilis simultaneously.Based on the blast results of D-LOOP gene of M.piceus,COI gene of C.idella,16SrRNA gene of H.molitrix and 12SrRNA gene of H.nobilis,four pairs of specific primers were designed to for the four fish different gene sequences.The identification method of four fish was established by single PCR and multiple PCR.The optimal conditions for PCR were explored by orthogonal experimental design L9(34).The results showed that the size of anticipated PCR products for the target gene of M.piceus,C.idella,H.molitrix and H.nobilis were 298、210、306 and 639 bp respectively.Under the optimized conditions,the method has a high specificity,and the 4 fish target gene in plasmid could be amplified simultaneously with clear bands at the amount of template lower to 50 pg.Furthermore,the method was stable by verifying in simulated examination and marketing examination.The study concluded that a rapid,specific and sensitive multiplex PCR method was established for identifying and discriminating fish products compositions from M.piceus,C.idella,H.molitrix and H.nobilis.