Rapid identification of Takifugu genus using visual loop-mediated isothermal amplification

Some Takifugu species are commonly found in the coastal areas of China, Japan, Thailand, and Korea and cause pufferfish poisoning, which is toxic and even lethal to humans. From 2010 to 2015, there were 430 cases of pufferfish poisoning worldwide, resulting in 52 deaths. Identification of Takifugu species is imperative to reduce financial losses and ensure food safety. Here, visual loop-mediated isothermal amplification (LAMP) was applied to identify Takifugu species. Conserved regions within the mitochondrial DNA among different Takifugu species were selected to design LAMP primers. In 55 min of amplification, sufficient DNA was obtained to observe the results with the naked eye, without the need for complicated instruments. The method was highly specific, with no cross-detection of 17 other fish species, namely, 7 Tetraodontiformes species and 10 commercially important fish. The method showed a detection limit of 0.1 ng Takifugu DNA and was successfully validated to detect Takifugu in cooked fish and the vomitus of poisoned patients. This rapid and visual LAMP method is a useful tool to prevent false labeling, protect consumer rights, and reduce the risk of pufferfish poisoning. PRACTICAL APPLICATION: The loop-mediated isothermal amplification method established in this study can identify cooked or digested fish products containing 1% or more of Takifugu. Therefore, it can be used for the visual detection of Takifugu products and the medical diagnosis of Takifugu poisoning.