摘要
[目的]应对出口水产品国外技术贸易壁垒,建立一种能被国际上普遍接受的进出口水产品中斑点叉尾鮰物种真伪鉴定方法,该方法也有助于我国水产品行业商业欺诈认定裁决工作。[方法]采用酚-氯仿抽提法提取水产品中基因组DNA,利用自行设计的引物D-loop F/D-loopR,对斑点叉尾鮰及大口鲇、革胡子鲶、鲤和鲫等物种的线粒体DNA D-loop和COⅠ区进行PCR扩增测序比对,根据斑点叉尾鮰物种D-loop区的序列寻找其特异性酶切位点,对D-loop区进行酶切鉴定。[结果]]建立的DNA提取技术可以满足水产品中线粒体D-loop扩增的要求,斑点叉尾鮰D-loop对应的最佳退火温度为54.8℃,其在D-loop序列的200 bp处有一特异性酶切位点EcoRⅠ。[结论]建立的斑点叉尾鮰产品中DNA提取、扩增技术及EcoRⅠ特异性酶切图谱方法,不需测序就能够将斑点叉尾鮰与其他近似物种区分开来,该方法适于进出口水产品贸易中斑点叉尾鮰产品的物种真伪鉴定。
[ Objective ] To deal with technical barriers to the international aquatic trade, the channel catfish species identification methods should be established in processed aquatic products. It is also helpful to commercial fraud identification and adjudication in aquatic products domestic trade . [Method]This research extracts DNA in aquatic semi-finished products by phenol - chloroform extraction , and makes sequence alignment on PCR amplification using the primers D-loopF/D-loopR designed to mitochondrial DNA D-loop and COl region of lctalurus punctatus, Silurus meridionalis, Clarias leather, Cyprinus carpio and Carassius auratus. According to the D-loop sequence of channel catfish species ,we try to get a specific restriction site to identifiy the species by D-loop area enzyme digestion. [ Result ] The DNA extraction technology established in this study can meet the processing aquatic products in the mitochondrial D - loop amplification, and the suitable annealing temperature corresponds to D-loop was 54.8 ℃, and a specific enzyme loci EcoR I（200 bp） was found successfuly to identifiy lctalurus punctatus. [Conclusion]The methods of DNA extraction and rapid molecular species identification in the processed channel catfish products could distinguish this species accurately from others without sequencing and be applied to species authentication of the processed products of Ictalurs punctatus in the import and export trade.
出处
《安徽农业科学》
CAS
2015年第11期20-23,共4页
Journal of Anhui Agricultural Sciences
基金
国家质检总局资助项目(2007IK015)
关键词
斑点叉尾鮰
物种鉴定
线粒体DNA
Ictalurus punctatus
Channel catfish
Speieces identification
Mitochondrial DNA sequence
