Identification of Puffer Fish of the Genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, Using Multiplex PCR

Abstract

A multiplex PCR assay was developed to identify widespread poison-containing puffer fishes of the genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, using a novel KUGEN_ILSspec primer set which is specific for 12S ribosomal RNA (12S rRNA) and Cytochrome b (Cyt b) genes. KUGEN_ILSspec set is composed of fish positive-amplified primers that produced a 289-bp fragment while L. lunaris-, L. spadiceus- and L. inermis-specific primers produced 123-, 196- and 493-bp fragments, respectively. The sensitivity of this primer set was found to be high as it was capable of amplifying targeted DNA at concentrations as low as 1 ng/μL. Moreover, the primer set was shown to amplify intact DNA and species-specific fragments from heat-processed food. Consequently, multiplex PCR technique in combination with KUGEN_ILSspec primer set could offer an effective measure for detecting poisonous puffer fish contamination in both fresh and processed fish products, which will greatly aid in public health control and law enforcing endeavors.