Rapid detection of Arothron species by real-time fluorescence and colorimetric loop-mediated isothermal amplification assays targeting the mitochondrial cytochrome b gene

Abstract

The pufferfish-poisoning epidemic is caused due to many consumers blindly tasting fish of an unknown species or adulterating food ingredients. Arothron is a pufferfish containing tetrodotoxin and saxitoxin in their bodies. Poisoning incidents of caused by Arothron occur occasionally. The purpose of the current study was to develop a simple identification method for Arothron fish based on loop-mediated isothermal amplification (LAMP) using visual colorimetric and real-time fluorescence measurements. The specificity and sensitivity of the techniques were evaluated, and the results proved that identification method was highly specific, with no cross-reaction occurring in non-Arothron fishes. The sensitivity was as low as 1 ng/L in visual colorimetric and real-time fluorescence LAMP assays. Arothron was used to simulate cooked goods and digested vomit to test further if it met the requirements of rapid detection in commercial use and medical testing. The feasibility results proved that the samples could also be successfully detected after cooking or digestion. Real-time LAMP is rapid and colorimetric LAMP is visible to the naked eye. Due to these characteristics, the LAMP method is expected to become an effective tool for species identification to meet the growing demand for food safety control of fish products.