A novel semi-multiplex PCR assay for identification of tropical eels of genus Anguilla in Indonesian waters

摘要

A one-step semi-multiplex polymerase chain reaction (PCR) is proposed to distinguish seven species and subspecies of tropical eels, i.e., Anguilla celebesensis, A. interioris, A. nebulosa nebulosa, A. marmorata, A. borneensis, A. bicolor bicolor, and A. bicolor pacifica, known in Indonesian waters. Seven pairs of species-specific primers, including two forward and seven reverse sequences, were designed after alignment of published and new complete mitochondrial cytochrome b (1,140 bp) and 16S ribosomal RNA (rRNA) (1,120 bp) sequences. All species-specific primer pairs are included in one PCR mix, but only one pair of them can amplify a specific fragment from the template DNA that is analyzed. The semi-multiplex PCR amplified a fragment of 230 bp for A. b. bicolor, 372 bp for A. n. nebulosa, 450 bp for A. borneensis, 620 bp for A. marmorata, 670 bp for A. b. pacifica, 720 bp for A. celebesensis, and 795 bp for A. interioris, which are then separated by DNA agarose gel electrophoresis.