Development of a fast real-time PCR assay based on TaqMan probe for identification of edible rice grasshopper (Oxya chinensis) in processed food products

Abstract

Interest in using insects as an alternative source of food for humans is increasing. However, few analytical methods provide accurate information about the presence of insect species in processed foods. In this study, we developed a fast real-time PCR assay based on a TaqMan probe that can be performed within 40 min to detect edible rice grasshopper in commercial food products. A rice grasshopper-specific primer pair and probe targeting the cytochrome c oxidase subunit 1 (COI) gene were newly designed, having an amplicon size of 110 bp. The specificity of this primer pair and probe was verified using 19 insects and five crustaceans and no cross-reactivity was obtained against the non-target species. The absolute limit of detection (LOD) was 0.5 pg of rice grasshopper DNA, and as low as 0.1% of rice grasshopper was detected in raw, heat-treated, and autoclaved binary insect mixtures. To evaluate the effect of food matrix, binary mixtures containing rice grasshopper in wheat were used additionally, and at least 0.1% of target species was detected using this assay. The applicability of this assay was confirmed using nine commercial food samples labeled as containing rice grasshopper or locust. The fast real-time PCR developed in this study is a specific and sensitive method for identifying edible rice grasshopper in various food samples.