AUTHENTICATION OF PRODUCTS FROM SNAPPER USING DNA BASED TECHNIQUES
Loading...
Date
2018
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Dr. J. Jayalalithaa Fisheries University
Abstract
Seafood adulteration is an important issue for food authorities, because species substitution and mislabeling present the major commercial frauds in seafood market sector around the world. In this study, different PCR based methods such as PCR-RFLP, PCR-SSCP, PCR-FINS and PCR-AFLP protocols were developed to authenticate nine different snapper species viz., Lutjanus fulvus, L. gibbus, L. lemniscatus, L. argentimaculatus, L. rivulatus, L. quinquelineatus, L. fulviflamma, L. madras and L. decussatus of commercial importance in the region of Thoothukudi, South India. For authentication analysis, fresh snappers and three processed snapper products such as frozen, cooked and fried snappers were used. Four different mitochondrial genome regions such as cytochrome b, 12S rRNA, 16S rRNA and hyper variable D-loop region were chosen as the molecular targets for authentication. Four sets of primers viz., Fish-CytB-F/Fish-CytB-R, 16S-SGS-F/16S-SGS-R, 12SU-F/12SU-R and Fish-DL-F/Fish-DL-R to amplify cytb, 16S rRNA, 12S rRNA and D-loop regions,respectively were designed using bioinformatics tools such as NCBI GenBank database, CLUSTAL OMEGA and BioEdit. The PCR amplified the respective targets successfully and the amplicons were sequenced, analysed using BLAST tool in NCBI and 24 sequences of different species of South Indian coast were deposited in GenBank database. All the PCR amplicons were used for developing a suitable PCR-RFLP protocol to authenticate nine snapper species. Webcutter software was used for in silico analysis to choose the suitable restriction enzymes for the respective mitochondrial region. Suitable restriction enzymes selected were Cfr13I, HaeIII and Tsp509I for cyt b region, Mn1I, MspAI and Sau3AI for 16S rRNA, BseDI, Mn1I and MspAI for 12S rRNA and Tsp509I for D-loop region. PCR-RFLP performed with these restriction enzymes on the four regions showed that cytb could differentiate only two snappers viz., L. rivulatus and L. decussatus with the enzyme, Cfr13I; and L. madras and L. fulvus from others with the enzyme, HaeIII. The 16S rRNA region differentiated only L. quinquelineatus from other five species with the enzyme, Mn1I while Sau3AI differentiated L. rivulatus from other species. The 12S rRNA region differentiated L. madras, L. quinquelineatus and L. decussatus from the other snapper species with the enzyme, BseDI. But, PCR-RFLP was very successful with D-loop region that differentiated all the nine species with a single enzyme, Tsp509I. The PCR-RFLP pattern obtained for the frozen, cooked and fried snappers were also similar to that of fresh snappers proving that the protocol was suitable for processed products.
PCR-SSCP performed on the target regions viz., cytb, 12S rRNA and 16S rRNA could not differentiate all the snapper species; but few species were differentiated randomly. On the other hand, SSCP of D-loop region differentiated all the nine snapper species with the newly designed reverse primer, DLFR for the forward primer, Fish-DL-F that produced a 360 bp D-loop fragment. PCR-FINS was used to construct the phylogenetic trees separately for 12S rRNA and D-loop sequences of snapper species using the Neighbor-Joining method. All the snapper species got placed in separate clusters with bootstraps values from 51 to 100, enabling differentiation by the 12S rRNA and D-loop region sequences without any ambiguity. PCR-AFLP developed using single pre-selective primer, EcoRI, and three selective primers viz., ACC, ACG and ACT also differentiated all the nine snapper species yielding specific AFLP markers that can further be used to develop or study unique regions of individual snapper species of importance.
In this study, among the four DNA based methods viz., PCR-RFLP, PCR-SSCP, PCR-FINS and PCR-AFLP tried for the authentication of nine processed snapper products, PCR-SSCP, PCR-FINS and PCR-AFLP methods could differentiate all the snapper species. However, these methods are not widely employed due to various difficulties in field application. For instance, FINS needs an expensive DNA sequencer for routine sequencing, while AFLP involves complicated procedures besides giving high inter and intra-species variation posing difficulty in the interpretation of the results. Likewise, PCR-SSCP also requires skilled persons due to stringent procedure in running electrophoresis. Hence, PCR-RFLP is a better choice for adaptation by the regulatory agencies. In this study, PCR-RFLP protocol targeting D-loop region of mitochondria was found more reliable by means of repeatability and low cost in authentication of fresh as well as the processed snapper products within 8 h, thereby add on to its suitability for regulatory purposes.
Description
Keywords
null