The aim of this proof-of-principle study (open access) was to design a universal DNA microarray (“DNA Chip”) to distinguish all edible fish species by comparing hybridization signal patterns from samples with patterns obtained from reference specimens.
The researchers designed a universal set of 96 DNA probes that cover all fish species of food interest. These were narrowed down by virtual modelling experiments from a long-list of 28,000 candidates which they had generated experimentally. They also included 4 control probes (sequences not present in edible fish). All probes were based on sequences from either 16S ribosomal RNA or cytochrome b.
DNA was isolated with either a CTAB method or with commercial DNA extraction kits. The gene markers cytb (approx. 464 bp) and 16S rDNA (approx. 600 bp) and an additional pUC57 vector DNA region (542 bp) were amplified in triplex PCRs. The DNA probes were spotted contactless using piezoelectric dispensing technology as 19 × 19 arrays. For hybridization of the generated PCR amplicons on the prepared microarrays the INTER-ARRAY Hybridization Kit was used according to manufacturer's specifications. The arrays were measured directly after staining and then processed using the INTER-VISION GENOTYPING 1.2.0 software.
The authors tested 86 fish fillets sourced from verified suppliers and were able to correctly identify all species by hierarchical clustering analysis of the results. The entire process takes a few hours. They conclude that the method is ready for further validation and ruggedness testing. More replicates and species should be analyzed to confirm current results. Likewise, the robustness of the DNA array should be determined, e. g. by using different thermocycler or users and laboratories.
Graphical abstract from the paper
