Discrimination method of seven species pufferfish of the genus Takifugu using DNA chromatography

Identifying poisonous plants and animals is very important because they not only can cause food poisoning, but also can be eaten for the purpose of suicide. A pufferfish is a poisonous fish that contains tetrodotoxin. In Japan, 136 pufferfish poisoning cases occurred from 2015 to 2019, but in many cases, the specific species involved was unidentified. To address this, we focused on a rapid and simple DNA chromatography technology called Single-stranded Tag Hybridization (STH). We collected seven pufferfish species of the genus Takifugu and designed species-specific primers as target regions of cytochrome c oxidase subunit I (COI) and cytochrome b with a specific base sequence at the 3' end. STH-PCR was performed in two separate reactions. After mixing the PCR products, a developing solution was added to perform chromatograph development and the results were visually analyzed. Specific lines were detected in all seven species. The pufferfish species could be properly determined using between 0.1 ng and 50 ng of template DNA. The PCR product length was 85-149 bp, making it very resistant to degradation. The species could be properly identified even in a mixture of multiple pufferfish species DNA. Furthermore, verification was performed using the supernatants of digested samples with artificial gastric juice and processed foods. Extracted DNA was obtained in all but the highly roasted fins, enabling discrimination. Overall, we applied a novel DNA chromatography detection system capable of discriminating seven species of the genus Takifugu, which have closely related DNA sequences.