盐干海参的2种核酸提取方法比对及其分子鉴定

Comparison of 2 kinds of nucleic acid extraction methods and molecular identification of salt dried sea cucumber

中文关键词:  盐干海参  核酸提取  分子鉴定

英文关键词:salt dried sea cucumber  nucleic acid extraction  molecular identification

中文摘要:

      目的 建立盐干海参的核酸提取及其分子鉴定方法。方法 选取5种盐干海参, 采用直接处理、洗涤处理和浸泡处理3种处理方式, 比较磁珠法和离心柱法两种核酸抽提方式的提取质量, 并采用聚合酶链式反应(polymerase chain reaction, PCR)法对提取的核酸进行分析确认。结果 同一种抽提方式不同海参处理组间核酸质量没有显著差异, 而两种抽提方式间核酸质量差异显著, 其中磁珠法提取的核酸得率高、纯度高, 而离心柱法提取的核酸得率及纯度均偏低。经PCR扩增验证, 磁珠法提取的核酸效果较佳。将扩增产物进行测序及系统进化分析发现5种海参实际种类与标注名称仿刺参存在较大差异。结论 盐干海参处理方式对核酸提取结果影响不大, 磁珠法提取核酸效果较好, 单纯依靠细胞色素c氧化酶亚基I (cytochrome c oxidase subunit I, COI)和16S rRNA基因通用引物并不能对所有的海参种类进行鉴定。

英文摘要:

      Objective To establish nucleic acid extraction and molecular identification methods of salt dried sea cucumber. Methods Five kinds of salt dried sea cucumbers were selected and treated by direct treatment, washing treatment and soaking treatment, the qualities of nucleic acid extracted by magnetic bead method and centrifugal column method were compared, polymerase chain reaction (PCR) was used to analyze and confirm the extracted nucleic acid. Results There was no significant difference in different sea cucumber processing group by the same extraction method, but there was significant difference in the quality of nucleic acid between the two extraction methods, among which the nucleic acid extracted by magnetic bead method had high yield and high purity, while the nucleic acid extracted by centrifugal column method had low yield and purity. PCR results verified that nucleic acid extracted by magnetic bead method was better. The amplified products were sequenced and phylogenetic analysis showed that the actual species of the 5 kinds of sea cucumbers were significantly different from their labeled names. Conclusion The treatment of salt dried sea cucumber has little effect on the results of nucleic acid extraction, while the magnetic bead method was better, only use the cytochrome c oxidase subunit I (COI) and 16S rRNA gene universal primers are not enough to identify all sea cucumber species.