Shrimps are the prime resource from both commercial fisheries and for aquaculture in many countries, account for more than 30% of global consumption of seafood Worldwide. Species identification using morphological criteria is difficult to non-specialist, and shell-less specimens, processed products or larval and juvenile forms are morphologically unidentifiable. Therefore, to enforce labeling regulations and prevent product substitution, there is a need for sensitive analytical methods that can be used to determine the species of a seafood product with no detectable external features. This study describes a multiplex PCR assay with species-specific primers based on the 16S rRNA mitochondrial gene to identify the commercially important shrimp species such as; Fenneropenaes indicus, Penaeus monodon, P. semisulcatus, Litopenaeus vannamei, and fresh water prawn Macrobrachium rosenbergii. The regions which show maximum interspecific variations were selected through whole mitochondrial genome analysis and which paves a way to design five pairs of species-specific primers based on the 16S rRNA were developed for species identification. The sensitivity estimation indicated that the species-specific primers could correctly amplify the target 16S rRNA gene and which yield band sizes of 220, 376, 846, 275 and 750 bp respectively. The specificity of the primers was very high since it doesn't cross-react with any one of the closely related species under the same family. The unique band patterns were also obtained in processed shrimp products without any degradation or alteration in the major fragments. The proposed method was also validated with 66 shrimp products such as frozen, fried, cooked and canned shrimp products collected from all over the country.
This is the first time that directs species-specific multiplex PCR assy was developed and fully validated for rapid and economic simultaneous identification of five commonly consumed shrimp species. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefold by detecting five different species in a single assay platform. Thus, the developed protocol can be performed within 2 hrs to authenticate five shrimp products of commercial significance so it can be used to expose fraudulent substitution of processed shrimps in national and international trade.