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Comp Biochem Physiol Part D Genomics Proteomics. 2006 Mar;1(1):139-44. doi: 10.1016/j.cbd.2005.09.004. Epub 2005 Nov 9.

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene.

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Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan.


This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes - BanII, DdeI, and NlaIII - were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.


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