昆虫学报 ›› 2020, Vol. 63 ›› Issue (1): 97-103.doi: 10.16380/j.kcxb.2020.01.011

• 研究论文 • 上一篇    下一篇

通用引物双重PCR在节肢动物物种鉴定中的应用

王彦坤1, 李天楚2, 粘景梓1, 陈思聪1, 郝志霞1, 姜金壮1, 黄大卫1,2,*   

  1. (1. 河北大学生命科学学院, 河北保定 071002; 2. 南开大学生命科学学院, 天津 300071)
  • 出版日期:2020-01-20 发布日期:2020-01-19

Application of duplex PCR with universal primers in arthropod species identification

WANG Yan-Kun1, LI Tian-Chu2, NIAN Jing-Zi1, CHEN Si-Cong1, HAO Zhi-Xia1, JIANG Jin-Zhuang1, HUANG Da-Wei1,2,*   

  1.  (1. College of Life Sciences, Hebei University, Baoding, Hebei 071002, China; 2. College of Life Sciences, Nankai University, Tianjin 300071, China)
  • Online:2020-01-20 Published:2020-01-19

摘要: 【目的】本研究旨在使用基于线粒体基因通用引物的双重PCR技术同时扩增单一样本中两条标记基因,从而达到简化节肢动物物种鉴定流程的目的。【方法】在一次PCR实验中同时加入可扩增线粒体COI基因和16S rDNA两个不同分子标记的引物,对3纲8目14科的14种节肢动物物种标本的基因组DNA进行扩增;扩增产物经电泳和胶回收后测序,并BLAST在线搜索相似序列,验证基于通用引物的双重PCR在不同的动物类群中用于物种鉴定的有效性。【结果】应用基于COI和16S rDNA的引物从分属于3纲8目14科的14种节肢动物基因组DNA中均可成功扩增目的基因;扩增产物测序结果进一步证实了扩增的准确性。【结论】通过本方法进行物种的分子鉴定,不仅可以保证物种鉴定的高准确率,还可以明显减少时间与DNA样本量的消耗,这对需要快速准确鉴定物种或珍稀的材料样本十分重要。

关键词:  节肢动物, 物种鉴定, DNA条形码, 线粒体基因, 双重PCR, 通用引物

Abstract: 【Aim】 The objective of this study is to amplify two marker genes in a single sample by duplex PCR with universal primers of mitochondrial genes, so as to simplify the process of arthropod species identification. 【Methods】 The primers that could amplify two different molecular markers including mitochondrial COI gene and 16S rDNA were added in a single PCR experiment, and the genomic DNA of specimens of 14 arthropod species belonging to 14 families of eight orders of three classes were amplified. The amplification products were sequenced after electrophoresis and gel extraction, and the similarity sequences were searched using BLAST online, to test the effectiveness of the universal primers based on duplex PCR method for species identification among different animal groups. 【Results】 The primers based on COI and 16S rDNA could amplify target genes from the genomic DNA of the 14 species belonging to 14 families of eight orders of three classes. The sequencing results of the amplified products further confirmed the accuracy of the amplification. 【Conclusion】 This method not only ensures the high accuracy of species identification, but also significantly reduces the consumption of time and DNA sample, which is very important for the rapid and accurate identification of species, especially for rare material samples.

Key words: Arthropod, species identification, DNA barcoding, mitochondrial gene, duplex PCR, universal primers