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Using loop-mediated isothermal amplification for fast species delimitation in eels (genus Anguilla), with special reference to the European eel (Anguilla anguilla)

2019; Elsevier BV; Volume: 101; Linguagem: Inglês

10.1016/j.foodcont.2019.02.022

1873-7129

Gesche Spielmann, Sonja Ziegler, Gerhard Haszprunar, Ulrich Busch, Ingrid Huber, Melanie Pavlovic,

Fish Ecology and Management Studies

Fast, reliable and easy to handle methods are required to survey the introduction of foreign eel species into German waters, to detect incorrect labelling of food as well as for ecological field studies e.g. on board a ship. Several DNA-based methods are available for identification of the European eel (Anguilla anguilla). However, the need for expensive and bulky equipment is not convenient for all applications, particularly those related to field work. Therefore, loop-mediated isothermal amplification (LAMP) assays were developed for identification of A. anguilla and the genus Anguilla, respectively. The Anguilla genus LAMP assay was able to detect at least 500 pg DNA per reaction of all four tested Anguilla species, the European eel (A. anguilla), its closest relatives (A. rostrata and A. australis), and the Japanese eel (A. japonica). The detection limit for the A. anguilla specific assay was also 500 pg DNA per reaction and, in combination with a rapid simplified DNA extraction method, both assays were able to detect one single egg of the species A. anguilla per LAMP reaction. None of the two LAMP assays showed false positive results among 112 non-Anguilla species tested. The Anguilla genus-specific LAMP system detected all 34 Anguilla specimens examined, while the second LAMP only detected the 18 A. anguilla specimens, but not the 16 specimens classified as other Anguilla species. The total analysis time, including DNA extraction and data evaluation, was no more than 90 min per experiment. A further time reduction to 70 min is possible by shortening the amplification time and omitting the melting curve analysis without lose of critical information.