Rapid identification of pufferfish in roast fish fillet by real-time PCR

The purpose of this study was to develop a rapid method based on a real-time PCR assay designed to identify the presence of pufferfish in roasted fish fillet. Specific primers and probes were designed targeting Takifugu spp. and Lagocephalus spp., the most common genera in China. Specificity and sensitivity of this assay design were tested by using artificially spikes of pufferfish mixed in with other fish, such as Gadus and Thamnaconus septentrionalis,among others. Fifteen samples of retail roasted fish fillet and six samples from a 1999 poisoning event that occurred in Fujian province China were analysed for pufferfish. When the assay design was validated, no cross-reaction was observed between pufferfish and other species of fish. The limit of detection (LOD) was determined to be 0.001 ng pufferfish template, and the sensitivity of the method was 1%. Lagocephalus lunari was detected in six samples assayed from 1999 and no pufferfish was detected in the 15 retail roasted fish fillet samples tested. These results showed that the method was efficient for screening for pufferfish contamination in the roasted fish fillet and it could benefit public health protection by reducing the risk of tetrodotoxin poisoning.