Rapid On-Site Identification for Three Arcidae Species (Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii) Using Ultrafast PCR Combined with Direct DNA Extraction

Abstract

Granular ark (Tegillarca granosa), broughton's ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological characteristics of the three species, the less valuable species could be exploited for food fraud. In this study, we developed a rapid on-site identification method based on a microfluidic chip for the detection of the three ark shell species. We designed new species-specific primers, targeting the genes encoding mitochondrial cytochrome b or cytochrome c oxidase I, for the identification of the three ark shells and estimated their specificity against 17 species, which amplified only the target species. The sensitivity of each primer was 0.001 ng. In addition, this method was further improved to develop a direct ultrafast polymerase chain reaction (PCR) for on-site food monitoring, which would allow for completing the entire procedure (from sampling to obtaining the results) within 25 min without DNA extraction. Our direct, ultrafast PCR was successfully applied to differentiate the three species from 29 commercial products. Therefore, this assay could be used as a rapid and cost-effective approach for the on-site identification of ark shells in commercial food products.

Keywords: Anadara broughtonii; Anadara kagoshimensis; Tegillarca granosa; ark shell adulteration; direct ultrafast PCR; on-site identification.

Figure 1

Sequence alignment of the mitochondrial genomes. (A) Alignment of cytochrome b (cytb) region against 17 different shellfishes or seafood species and T. granosa-specific primer site (yellow sequences). (B) Alignment of cytochrome oxidase subunit I (COI) region against 17 different shellfishes or seafood species and A. broughtonii-specific primer site (green sequences). (C) Alignment of COI region against 17 different shellfishes or seafood species and A. kagoshimensis-specific primer region (blue sequences).

Figure 2

Standard curves by Ct values against DNA concentrations for (A) granular ark, (B) broughton’s ribbed ark, and (C) half-crenate ark. All samples were tested in triplicate.