| Surimi, a refined myofibrillar protein product, is high in protein and low in fat. It is made from raw fish through a series of processes. It has unique texture, delicious flavour and rich nutritional value. The global surimi industry has expanded in recent decades, but new challenges have emerged. As surimi is a source of myogenic fibrous protein derived from processed fish flesh, fish species identification through morphological and sensory methods is impossible. Moreover, the nutritional value and quality of surimi vary considerably depending on the fish species used to make surimi, so determining the species of fish used in surimi is essential for quality assurance. Additionally, whiteness and gel characteristics are key factors in determining surimi quality and grade. Improvements in these attributes increases surimi grade and market price. Black carp (Mylopharyngodon piceus), grass carp (Ctenopharyngodon idella), silver carp (Hypophthalmichthys molitrix), and bighead carp (Hypophthalmichthys nobilis) are the four major freshwater fish species in China, known for their low cost, white flesh, and effective gelatinization properties. Their use in surimi production can enhance whiteness and texture, increasing surimi’s grade and price. Therefore, driven by economic interests, some manufacturers add black carp, grass carp, silver carp, or bighead carp to surimi without labeling them, misleading consumers and disrupting market order. Therefore, developing an accurate, sensitive, and rapid method to identify these species in marine fish surimi is crucial.
Digital PCR is a third-generation PCR technology with higher specificity and sensitivity, allowing quantification of the DNA template of the sample to be tested without the use of a standard curve. Crystal digital PCR combines both microtitre and microarray technologies, enabling sample partitioning, PCR amplification and fluorescence analysis to be performed on a chip, minimising handling time and cross contamination. In this study, we designed a pair of specific primers/probes based on the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene for the simultaneous detection of four freshwater fishes, and developed a crystal digital PCR method. This method enabled the quantitative detection of black carp, grass carp, silver carp, and bighead carp in marine fish surimi. The reaction conditions were optimised via an orthogonal experimental design, and the method's specificity and sensitivity were subsequently validated. Fifty commercially available surimi products were tested to verify its practicality in detecting target species.
The results indicated that the method exhibited high specificity. Furthermore, amplification of sample fish DNA (0.2 ng/μL) generated copy numbers of 638.8, 402.5, 785.3, and 627.5 copies/μL for black carp, grass carp, silver carp, and bighead carp, respectively, with no cross-reactivity to 24 other fish species in the crystal digital reaction. The determination of the lower limit of quantification demonstrated that the method could detect sample DNA at a minimum concentration of 0.32 pg/μL for black carp, grass carp, and silver carp, and 0.064 pg/μL for bighead carp. Additionally, the method detected the minimum of copy number of 1.43 copies/μL for black carp, 2.87 copies/μL for grass carp, 2.69 copies/μL for silver carp, and 1.72 copies/μL for bighead carp. Among 50 commercial samples, 31 tested positive and 19 tested negative by dPCR analysis. Analyses of commercially available surimi samples demonstrated that the method could quantify copy numbers of components derived from black carp, grass carp, silver carp, and bighead carp. Furthermore, the qualitative results were consistent with those obtained through DNA barcoding technology. This study developed a sensitive and rapid crystal digital PCR method for quantifying ingredients derived from black carp, grass carp, silver carp, and bighead carp in marine fish surimi. This approach provided an effective technical reference for industry authorities and testing agencies to monitor the compliance labelling of freshwater fish components in marine fish surimi, ensuring transparency in surimi product authentication. |
