PCR-RFLP for authentication of different species of processed snappers using mitochondrial D-loop region by single enzyme

A PCR-RFLP protocol was developed for authenticating nine different snapper species by targeting the 515 bp fragment in highly polymorphic mitochondrial D-loop region with a newly designed primer Fish-DL-F/Fish-DL-R, followed by restriction digestion using a single enzyme, Tsp509I. Individual species could be differentiated by 3-5 major bands. Very closely related species like L. fulvus and L. fulviflamma gave similar pattern due to high (94%) identity, while the other seven species were clearly differentiated. The protocol was also found successful in distinguishing the species in frozen, cooked and fried snappers. This protocol was validated with snapper samples procured from the fish market of Thoothukudi, India. Hence, the method can be efficiently used to authenticate snapper species within 8 h by regulatory authorities to avoid seafood species substitution.