Novel real-time PCR protocol for the detection of house cricket (Acheta domesticus) in feed

A new real-time PCR protocol was developed for the detection of house cricket (Acheta domesticus) in feeds. A sequence of 97 bp size within the mitochondrial barcode region (cytochrome oxidase c subunit I - COI) was amplified by the chosen specific primer and probe set. The specificity of the PCR system was confirmed with a broad range of plant and animal species. The limit of detection with pure A. domesticus DNA diluted in aquaculture feed DNA was 1 genome copy, corresponding to an absolute amount of 2.14 pg DNA. In addition, the sensitivity was investigated in dependence on different processing treatments of the insect material. The method reliably detected pasteurized (80 °C, 10 min) and oven-dried (60 °C, 3 h) A. domesticus in feed mixtures down to 0.1% (w/w). Practical applicability of the proposed protocol was tested on 33 commercial feeds not declaring the presence of house cricket in the list of ingredients, and on two model feeds spiked in-house with A. domesticus. The method clearly recognized the presence of house cricket in model feeds down to 0.1%, a concentration residing in the range of accidental contamination rather than deliberate addition. It is concluded that the tested system is suitable for practical application, enabling the sensitive and reliable detection of A. domesticus material as a newly allowed component in aquaculture feed.