Overview

Motto: Research First

We focus in our research on fish parvalbumin gene(s), in particular on their intron sequences. In spite of exons, these stretches of DNA were not subject of unifying selection pressure, hence they differ in substantial way among various fish species. Therefore, they can be employed as useful platform for species differentiation.

We seek to define stretches in such genes that are most suitable for designing primers and/or TaqMan probes for PCR in endpoint format and qPCR. Our goal is to learn relations among ray-finned fishes governing pattern of distribution of parvalbumin gene paralogs and sequence their introns in each species.

We use cloning of segments of fish parvalbumin gene into plasmid vectors together with subsequent sequencing as one of employed tools among common bioinformatic approaches and characterizations from qPCR data.

Collaborations

Department of Zoology, Faculty of Science
Charles University
Prague
Czech Republic

Department of Biochemistry and Microbiology,
UCT Prague
Czech Republic

Matís ohf. / Icelandic Food and Biotech R&D
Vínlandsleið 12,
113 Reykjavík
Iceland

Department of microbiology, molecular biology and biotechnology / Food Research Institute
National Agricultural and Food Centre ,
Priemyselná 4
824 75 Bratislava,
Slovak Republic

We also collaborate with dynamic spin-off company Elisadevelopment, located in the building of the Institute. Currently, they expand their activities into field of molecular biology, in particular forensic applications of DNA sequence-based species determinations.

Research

Our research aims to detection methods for fish species determination based mainly on polymerase chain reaction – PCR

Degenerate primers

Degenerate primers differ from conventional ones by degenerate bases in some positions in their sequence. Degenerate base represents variety of more than one nucleotides at this position –for instance A + G. In reality, degenerate primer is mixture of number of variants of sequences number of which results from combinatorial rules.

Employment of degenerate primers enables us to amplify some stretch of the parvalbumin gene in fish species without knowing any piece of sequence of the gene. Degenerate primer is designed based on alignment of number of known variants of such stretch of parvalbumin gene among fishes, so there is high probability it will pair with the respective stretch also in fish of our interest, still devoid of necessary sequence information.

End-point PCR

In this „classical“ variant of PCR we analyse newly designed sets of primers in respect of their specificity to target fish species, selectivity in terms of non-reactivity to other fish species from panel of fish species representing whole spectrum of ray finned fishes. This PCR format is the gold standard of PCR, also the most useful for routine testing and screening.

Real-time PCR

Compared to End-point PCR, Real-time PCR brings one extra information: it can quantify the amount of analysed target (i.e. fragment of fish species specific gene) in the sample. TaqMan probe not only conveys the amount of the specific DNA stretch into detectable light signal, but it also acts as another selection threshold parallel to both primers.

Cloning

We use cloning into plasmid vectors as a powerful tool for further handling with DNA stretch of our interest. Amplification in bacteria scales the amount up to the level needed for sequencing or for preparation of recombinant calibrator. Sequencing of cloned fragments is more convenient in some aspects than sequencing of amplicons from PCR.

Recombinant DNA as calibrator

In Real-time PCR serial dilutions of isolated DNA can be employed for construction of calibration curve. In the same way the cloned fragment of parvalbumin gene can serve as calibrator. This modification enables development of defined assays based on Real-time PCR.

Phylogenetic analysis and in silico processing

As a result of evolution, there exist differences in sequence between particular parvalbumin genes among fish species. However, exon parts of these sequences are subject of selection pressure as any mutation that cripples the function of resulting protein ceases further destiny of such variant. On contrary, introns evolve without such constraint and therefore are quite diverse compared to exons.

In evolution of ray finned fishes extra amplification(s) besides of those at the stem of vertebrate line did happen. As a result, quite big number of gene copies of parvalbumin gene can be present in each species. This multiplicity could hamper the specificity of the assay if not taken into consideration at the stage of marker sequence selection. It is therefore critical to understand relations between members of this family of parvalbumin genes. In our research we match sequential data obtained from sequencing amplicons and cloned parvalbumin fragments with available phylogenetic data. Towards this end, we process all sequences we obtain by usual bioinformatic tools to produce data of quality enabling such matching and further decisions.

Deep insight into the landscape of parvalbumin gene variants distribution among each fish species as well as among the whole group of ray finned fishes turns out to be the most critical condition for all of the above mentioned research activities. That is why we currently focus our research attention to this sphere.

Selected publications

Akhatova, D. – Laknerová, I. – Zdeňková, K. – Ólafsdóttir, G. – Magnúsdóttir, S. – Piknová, Ľ. – Kýrová, V. – Lerch, Z. – Hanák, P.: International interlaboratory study on TaqMan real-time polymerase chain reaction authentication of black seabream (Spondyliosoma cantharus).  Journal of Food and Nutrition Research, 57, 2018, pp. 27-37

Laknerová, I. – Zdeňková, K. – Purkrtová, S.  – Piknová, Ľ. – Vyroubalová, Š. – Hanák, P.: 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. Journal of Food Quality, 37, 2014, pp. 429–436. DOI: 10.1111/jfq.12114.

Hanak, P. – Laknerova, I. – Svatora, M.: Second intron in the protein-coding region of the fish parvalbumin gene – a promising platform for polymerase chain reaction-based discrimination of fish meat of various species. Journal of Food and Nutrition Research, 51, 2012, pp. 81-88. ISSN: 1336-8672

Zdeňková, K. – Akhatova, D. – Fialová, E. – Krupa, O. – Kubica, L. – Lencová, S. – Demnerová, K. Detection of meat adulteration: Use of efficient and routine-suited multiplex polymerase chain reaction-based methods for species authentication and quantification in meat products. Journal of Food and Nutrition Research, 57, 2018, pp. 351-362 ISSN: 1336-8672

Akhatova D., Hanak P., Laknerova I., Zdenkova K., Demnerova K.: PCR-based fish meat authentication, Chemical Reactions in Foods VIII 2017; Prague, Czech republic, 15.-17. 02.2017

Akhatova D., Hanak P., Laknerová I., Lencová S., Zdeňková K., Demnerová K.; Fish species identification by PCR using parvalbumin gene as a platform, Belfast Summit on Global Food Integrity, Belfast, United Kingdom, 28-31.05.2018