A multiplex PCR assay for the identification of five commercially important Portunid crabs: Portunus pelagicus, P. gladiator, P. sanguinolentus, Charybdis natator, and C. feriatus

Abstract

ABSTRACTPortunid crabs of the genus Portunus: P. pelagicus, P. gladiator and P. sanguinolentus and the genus Charybdis: C. natator and C. feriatus are economically important species for many tropical countries. An ongoing problem in the crab industry is involved with food labelling or food fraud in which many food manufacturers may have substituted the cheaper crabmeat for the more expensive ones due to the difficulty of species identification after many steps of food processing. To solve this problem, a convenient and accurate multiplex PCR assay using a species-specific primer set KUGEN_PORTspec was developed based on the nucleotide variation of cytochrome b and 28S ribosomal RNA. The primer set specifically amplified fragments of 286, 348, 418, 124 and 481 bp in P. pelagicus, P. gladiator, P. sanguinolentus, C. natator and C. feriatus, respectively, as well as a 220 bp positive control fragment. Specificity and sensitivity tests showed consistency in product sizes and absence of cross-species amplification with 0.1 ng DNA template limit of detection. Validation of the multiplex PCR assay on crabmeat prepared by steaming, mixing, freezing and canning was done and visualized by either agarose gel electrophoresis or automated capillary electrophoresis. The results indicated that the multiplex PCR assay using KUGEN_PORTspec is an effective tool for portunid species identification from both high- and low-quality DNA obtained from processed food. This newly developed primer set is an effective tool for crabmeat species identification to be used in future food industry and consumer protection programs.