4种鳕鱼多重PCR检测方法建立

Establishment of Multiplex PCR Detection Methods for Four Cod Species

摘要:

为了建立可对鳕鱼DNA进行特异性检测的PCR方法，从GenBank数据库下载太平洋鳕、大西洋鳕、狭鳕、绿青鳕4种鳕鱼的突触素样蛋白（pantophysin Pan I）基因序列，并用Bioedit 7.0软件对上述不同鳕鱼的该基因碱基序列进行比较。根据引物设计的基本原则，选择了鳕鱼与其他鱼类碱基序列上差异位点较多片段，设计出太平洋鳕、大西洋鳕、狭鳕、绿青鳕PCR特异性引物。用该引物分别对从7种鳕鱼和14种非鳕鱼鱼类的肌肉、脏器组织中提取的DNA进行PCR扩增。实验将所设计的引物通过两种引物组合、三种引物组合以及四种引物组合，进行多重PCR试验，结果表明，大西洋鳕、绿青鳕、太平洋鳕和狭鳕四种鳕鱼分别出现597、392、266、527 bp大小的清晰条带，4种引物之间相互不干扰，具有显著特异性。该方法具有较高灵敏度，低至4 ng/μL混合样品仍可检出。

Abstract:

To establish a PCR method for specific cod DNA detection, DNA sequences of pantophysin (Pan I) of four cod species, including those of Pacific cod (Gadus macrocephalus), Atlantic cod (Gadus morhua), Atlantic pollock (Pollachius pollachius), and Saithe (Pollachius virens), were obtained first from GenBank. The base sequence of the DNA of these four cod species was compared using Bioedit 7.0. According to the basic principles of primer design, cod fragments showing more significant base sequence differences from those of other fish species were selected for specific PCR primer design for the above-mentioned four cod species. These primers were used in the PCR amplification of DNA extracted from muscles and organ tissues of seven cod and fourteen other fish species. Combinations of two, three, and four primers were adopted for multiplex PCR experiments. The results showed clear 597, 392, 266, and 527 bp bands for Pacific cod, Atlantic cod, Atlantic pollock, and Saithe, respectively. In addition, no interference between the four primers was noticed, they are thus highly specific. The described method is highly sensitive and provides accurate results even for mixed samples with a concentration as low as 4 ng/μL.