DEVELOPMENT OF PCR-RFLP METHOD FORIDENTIFICATIONOF FISH PRODUCTS FROM DIFFERENTSPECIES OF SARDINES
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Date
2013-08-03
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Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Fisheries University
Abstract
PCR-RFLP technique was developed for the identification of five different species of sardines viz. Sardinella longiceps, S. gibbosa, S. albella, S. fimbriata and S. sirm. Samples collected from fish landing centres of Thoothukudi, TamilNadu, India were divided into 5 lots. One of the lot served as raw control. Other four lots were subjected to different preservation and processing methods and designated as cooked, chilled, frozen, and salt-dried. DNA was extracted from the tissues and amplified for the mitochondrial cytochrome b (mt cyt b) gene by using C-CB28dFand C-CB431R primer. The sardine specific gene got amplified at 147 bp. The amplified DNA fragment was then subjected to restriction digestion by using the enzymes, HinfI and MnlI. The enzymes cut the DNA fragment at their respective restriction sites to give different band patterns.
PCR-RFLP band patterns of different species of raw sardines showed that there were major bands in four species, viz , S. gibbosa, S. albella, S. fimbriata and S. sirm at different base pairs. There was no major band in S.longiceps which easily distinguishes it from other species. S.gibbosa had a single broad major band at 107 bp and no band above 50 bp. S.albella showed two clear bands at 107 bp and 80 bp; apart from a minor band at 55 bp. S.fimbriata had two bands, among which, the major band was at 107 bp. S.sirm also showed two bands, but the major band was at 75 bp, which distinguishes it from S.albella. PCR-RFLP patterns of the different species of cooked, chilled, frozen and salt-dried sardines have exhibited the same pattern as that noticed with raw sardines; in respect of the major bands and their molecular sizes. Although the minor band at 55 bp was absent in few species of sardines, it did not significantly affect the species differentiation. The specific banding pattern observed for the different species of processed sardines, thus make PCR-RFLP as a suitable technique for authentication of sardine species on commercial scale, even for processed products.
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