Interlaboratory Identification of Black Seabream (Spondyliosoma cantharus) as a Model Species on Basis of Polymerase Chain Reaction Targeting the Second Intron of the Parvalbumin Gene

Abstract

An end‐point polymerase chain reaction (PCR) targeting the second intron in the protein‐coding region of the parvalbumin gene of black seabream (Spondyliosoma cantharus) was used to identify this fish species. The reproducibility of the method was tested by a collaborative study in which four laboratories participated. Twenty‐eight samples of isolated DNA from fish meat were tested in participating laboratories by the presented identification method. Nine samples were from black seabream and the remaining 19 were fish flesh from the reference panel. In parallel, six frozen samples of fish meat, where three were of black seabream and remaining three from the reference panel, were tested in the laboratories by this method after various DNA isolation procedures. The PCR‐based method for species determination of black seabream presented in this study proved to be a reliable and independent on method of DNA isolation from meat among the isolation techniques used. Practical Applications This work deals with the interlaboratory assessment of a previously described end‐point polymerase chain reaction‐based method for fish species determination from fish flesh devoid of anatomical traits. The end‐point form of the test is simple enough to be broadly used by laboratories in the field. This novel approach based on species‐independent degenerate primers makes it possible, through the use of obtained amplicons, to design species‐specific primers, such as the ones tested here, in a quite straightforward manner. Therefore, the presented assay represents the first practical employment of a broader and more versatile approach capable of reacting in a relatively short time to fish species first emerging as new commodities on the market.

Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment

“…Furthermore, a pair of universal primers for pike DNA was designed; the length of the amplicon was approximately 412 bp (425 bp for E. niger). As the second intron of the parvalbumin gene appears to be a promising platform for fish species identification, 41,42 targeted region ranged from the 3′ end of the first intron to the 5′ end of the third exon of the parvalbumin gene (Figure 2). This approach allows the amplification of a large part of the nucleotide sequence encoding the EF-hand motif in the parvalbumin protein, including binding sites for calcium ions in E. lucius (Figure 3).…”

Twenty‐three years of PCR‐based seafood authentication assay development: What have we learned?

“…As mentioned previously, highly processed samples (thermal or high pressure treated) may exhibit low DNA integrity; as such, it is important that DNA-based assays can detect small fragment sizes (<200 bp). Many of the conventional PCR assays reviewed amplified DNA target regions <200 bp (Acar et al, 2017;Hellberg et al, 2010;Kang, 2019;Laknerová et al, 2014;Lin & Hwang, 2008a;Xiong et al, 2020). Xiong et al (2020) assessed the sensitivity of conventional PCR.…”

Testing of the Effect of Food Additives on the DNA Isolation from Mackerel and Fish Products

“…Získané amplikony o délce cca 600 bp úspěšně sekvenovali, což dokazuje vhodnost metody využívající CTAB pro následné analýzy DNA. Izolace DNA metodou využívající CTAB s následnou amplifikací části parvalbuminového genu byla úspěšně použita také pro identifikaci DNA mořana tmavého (Spondyliosoma cantharus)27 . Laknerová a spol 27.…”

“…Izolace DNA metodou využívající CTAB s následnou amplifikací části parvalbuminového genu byla úspěšně použita také pro identifikaci DNA mořana tmavého (Spondyliosoma cantharus)27 . Laknerová a spol 27. uvádějí, že kvalita i kvantita izolované DNA metodou využívající CTAB byla srovnatelná s DNA získanou komerčními kity na bázi silikátových centrifugačních kolonek.…”