Differentiation of the genus Anguilla is important for phylogenetic studies, ecological monitoring, and food forensics. Although various methods have been developed to identify these species, current methods are time-consuming and limit the number of species that can be detected. The aim of this study was to develop a rapid and convenient method to identify the genus Anguilla. To differentiate among seven commercially important species of Anguilla (A. japonica, A. anguilla, A. rostrata, A. celebensis, A. pacifica, A. marmorata, and A. bicolor pacifica), fluorescence melting curve analysis (FMCA) with peptide nucleic acid (PNA) probes were used. One primer set was designed to amplify the mitochondrial cytochrome b gene, and seven PNA probes were designed to identify each species from the cyt b gene. The PNA probes were labeled with four different fluorescent reporter dyes (FAM, HEX, Texas Red, Cy5) for multiplex analysis using two tubes. Based on FMCA, each PNA probe showed the highest Tm value when it was 100% complementary to the target sequence. In conclusion, the PNA-based FMCA method is suitable for differentiating among seven species of Anguilla and should be applicable in various fields, such as global marketing and scientific research.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Denny, S.K., Denny, A. & Paul, T. Distribution, prevalence and intensity of Anguillicoloides in the American eel, Anguilla rostrata, in the bras d’Or Lakes, Nova Scotia. BioInvasions Records 12, 19–26 (2013).
Sang, T.K., Chang, H.Y., Chen, C.T. & Hui, C.F. Population structure of the Japanese eel, Anguilla japonica. Mol. Biol. Evol. 11, 250–260 (1994).
Henkel, C.V. et al. First draft genome sequence of the Japanese eel, Anguilla japonica. Gene 511, 195–201 (2012).
Tzeng, W.N. et al. Identification and growth rates comparison of divergent migratory contingents of Japanese eel (Anguilla japonica). Aquaculture 216, 77–86 (2003).
Lee, N.S. et al. Morphological and molecular identification of a tropical glass eels Anguilla marmorata and A. bicolor pacifica from Philippines Coast. J. Fish. Mar. Sci. Edu. 27, 1109–1117 (2015).
Trautner, J. Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction. Inf. Fischereiforsch. 53, 49–51 (2006).
Slawomir, K., Remigiusz, P. & Jolanta, K. Eel species identification by polymerase chain reaction followed by restriction fragment length polymorphism (PCRRFLP). Med. Wet. 65, 315–318 (2009).
Ege, V. A revision of the genus Anguilla Shaw: a systematic, phylogenetic and geographical study. Dana Rep. 16, 1–256 (1939).
Watanabe, S., Aoyama, J. & Tsukamoto, K. Reexamination of Ege’s (1939) use of taxonomic characters of the genus Anguilla. Bull. Mar. Sci. 74, 337–351 (2004).
Watanabe, S., Aoyama, J., Nishida, M. & Tsukamoto, K. A Molecular genetic evaluation of the taxonomy of eels of the genus Anguilla (Pisces: Anguilliformes). Bull. Mar. Sci. 76, 675–690 (2005).
Alberts, B. et al. Molecular biology of the cell. New York and London: Garland Publishing Inc. (1994).
Gil, L.A. PCR-based methods for fish and fishery products authentication. Trends Food Sci. Technol. 18, 558–566 (2007).
Kim, J.W. et al. Comparison of PNA probe-based real-time PCR and Cobas TaqMan MTB for detection of MTBC. BioChip J. 7, 85–88 (2013).
Fiandaca, M.J., Hyldig-Nielsen, J.J., Gildea, B.D. & Coull, J.M. Self-reporting PNA/DNA primer for PCR analysis. Genome Res. 11, 609–613 (2001).
Rasmussen, R.S. & Morrissey, M.T. DNA-based methods for the identification of commercial fish and seafood species. Compr. Rev. Food Sci. F. 7, 280–295 (2008).
Berry, O. & Sarre, S.D. Gel-free species identification using melt-curve analysis. Mol Ecol Notes 7, 1–4 (2007).
Syvänen, A.C. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2, 930–942 (2001).
Deokhwe, H. et al. Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis. Biol. Proced. Online 17, -015-0027-0025. eCollection (2015).
Kim, E.M. et al. Easy method for discriminating the origins of manila clam Ruditapes philippinarum with a dual-labelled PNA-probe-based melting curve analysis. BioChip J. 9, 247–258 (2015).
About this article
Cite this article
Noh, E.S., Kang, H.S., Kim, E.M. et al. Rapid Differentiation of Seven Species of Anguilla Using PNA Clamping-based Asymmetric PCR with Fluorescence Melting Curve Analysis. BioChip J 12, 46–51 (2018). https://doi.org/10.1007/s13206-017-2106-y
- Fluorescence melting curve analysis
- Real-time PCR
- Species identification
- Cytochrome b