双重PCR熔解曲线峰快速检测食品中猪源性成分方法的建立

Establishment of duplex PCR melting curve for rapid identification of swine origins in foodstuff

摘要: 基于国际生命条码联盟(CBOL,the Consortium for the Barcode of Life)提出的barcoding技术所确定的序列区域,针对猪的线粒体COⅠ基因序列设计特异性引物。为了避免食品中PCR抑制剂的影响,本实验设置GCG(胰岛素受体)基因125bp的纯化片段为内参照,控制假阴性结果的出现。通过优化PCR反应条件,猪和GCG基因的特异性产物在79.2℃和75℃有特异性熔解峰。设置牛、羊、鸡、兔、鼠和空白对照,均无扩增。测序结果表明,该猪特异性引物的PCR产物含有245bp的核苷酸序列与GenBank中相应序列吻合;该方法检出限为0.001%。

Abstract: Primers specific to swine was designed for species-specific COⅠgene-amplification from barcoding was proposed by CBOL. Simultaneously in order to prevent false negative results, GCG gene fragment was used for the internal positive control (IPC) . Gene products of swine and GCG were represented in two melting peaks generated simultaneously at temperatures of 79.2℃ and 75℃ respectively. The specificity of the method was evaluated using template DNAs from bovine, chevon, chicken, rabbit, rat and NTC, whereas only amplification products of GCG gene were obtained. DNA sequencing results showed that the sequence of swine-specific products was 245bp, and which were 100% matching the corresponding sequence in the GenBank. The LOD of this method was 0.001%.