A SINGLE RESTRICTION ENZYME BASED RFLP (PCR-RFLP) TECHNIQUE FOR THE SPECIES DETERMINATION OF FIVE IMPORTANT LOACHES OF WESTERN GHATS USING COI GENE

Fish food substitution has become an important threat in both domestic and international  markets due to increasing international trade, per capita consumption, poaching etc. DNA based technique can be applied to identify  the species in any form including embryonic stages or any part of the fish or any form of processed fish food. In the present study, a simple method involved a single restriction enzyme based polymerase chain reaction,i.e., Polymerase Chain Reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) method was developed as a tool to prevent commercial frauds in fish trade and for determination of the five important loaches of Western Ghats to avoid substitution of cheaper fish for more expensive loach species when the usual identifying characteristics are removed. The Western Ghats loaches includes Lepidocephalus thermalis (Cobitidae), Nemachelilus triangularis, N.guentheri, N.semiarmatus and Bhavania australis (Balitoridae). The PCR products were digested with single restriction enzyme-Eco RII to identify five fish species of loaches includes Lepidocephalus thermalis (Cobitidae), Nemachelilus triangularis, N.guentheri, N.semiarmatus and Bhavania australis (Balitoridae).

The fishes were collected from various locations of Western Ghats -Tamil Nadu and Karnataka State.  DNA was extracted from the fish tissue and purity of DNA was analyzed with UV spectrophotometer at 260mm and 280 mm, the purity recorded was 1.60 -2.0 (Quality of DNA  indicated as good). DNA amplification was carried out by using the primer of Cytochrome C oxidase subunit 1 (COI) gene for Polymerase Chain Reaction (PCR) (Fig.1). The PCR products were digested with 1µl (10U/ml), 18 µl  of nuclease free water and 2 µl of 10X buffer. The restriction enzyme digestion was carried out by incubating at 37^oC for 16h.Finally the digested samples were separated and confirmed with 10% Polyacrylamide gel electrophoresis by using DNA markers. Then gel was documented and PCR-RFLP was analyzed for single restriction endonuclease, Eco RII, and it was able to distinguish between the five fish species of loaches, all five species could be differentiated (Fig.2). Thus, this method can be used to determine fish food fraudulents and also conserve the species.