International interlaboratory study on TaqMan real-time polymerase chain reaction authentication of black seabream (Spondyliosoma cantharus)
Abstract
An interlaboratory study of five laboratories testing real-time polymerase chain reaction (PCR) with TaqMan probe detection of black seabream (Spondyliosoma cantharus) fish is presented in this work. The method is oriented to the intron of the nuclear gene encoding the main protein of the musculature of the fish, parvalbumin.
This feature distinguishes the employed approach from those based on mitochondrial genes. Here, the intron is flanked by exon stretches highly conserved among species.
This provides a unique advantage when a new fish species emerges as a commodity on the market: Species-versatile degenerate primers can be easily designed on these conserved exon stretches. Therefore, the initial uncertainty of the species-specific sequence of the intron can, in such case, be bypassed during the adoption of the method to this particular new species, because the amplicon obtained at this pilot stage provides the sequence of the intron itself.
DNA isolates from eight exemplars of S. cantharus and from nineteen other fish species, the latter being used as negative controls, were tested in this study by participating laboratories. The readouts in qualitative assessment were 100% accurate.
The quantitative results provided an average value and variation among samples representing particular exemplars of S. cantharus.