Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96-100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers' health and economic interests.