A single enzyme PCR-RFLP protocol targeting 16S rRNA/tRNAval region to authenticate four commercially important shrimp species in India

Food authenticity is an issue of major concern for food authorities, as mislabeling represents one of the major commercial frauds. In this study, a novel PCR-RFLP protocol was developed as a tool to authenticate four shrimp products of commercial importance belonging to the family, Penaeidae, viz. Litopenaeus vannamei, Penaeus monodon, P. semisulcatus and Fenneropenaeus indicus. PCR amplification was performed targeting 16S rRNA/tRNAval region having an amplicon size of 530 bp using the specific primers for shrimps, 16S-Cru4/16S-Cru3. Subsequent restriction analysis with a single restriction enzyme, Tsp5091, yielded distinct RFLP pattern for each species of shrimps having fragment sizes below 150 bp. The unique RFLP patterns were also obtained in processed shrimp products without any degradation or alteration in the major fragments. The method was also validated with commercial shrimp products. Thus, the developed protocol can be performed within 8 h using a single enzyme to authenticate four shrimp products of commercial significance.