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