Defining the parameters of comet assay for reliably detecting DNA damage as a marker of X-ray based irradiation in insects treated with non-lethal doses for export fruit sanitation

<b>BACKGROUND</b>: Phytosanitary irradiation of fresh produce is used to prevent insect pests from spreading through trade pathways. Regulations stipulate minimum absorbed doses to ensure that pests are rendered inviable, though mortality is not a requirement. Consequently, live insects can sometimes be found in treated produce, but distinguishing them as having been irradiated is challenging. Currently, there is no simple, fast test that can simultaneously assess both insect and fresh produce tissues for irradiation treatment. The comet assay, which detects single strand DNA breaks as a marker of irradiation damage, is widely used in various applications but has not been adapted for detecting irradiated insects in imported fresh produce. This study aimed to define the essential parameters for applying the comet assay to both insect and fruit tissues.<b>RESULTS</b>: Using houseflies, clover root weevils, and mangoes as model systems, we evaluated the assay's effectiveness in detecting DNA damage caused by X-ray irradiation. Our findings indicate that the comet assay can reliably detect irradiation-induced DNA damage in both insects and fruit, with the assay parameters requiring careful adjustment to account for differences in tissue types and life stages of insects.<b>CONCLUSION</b>: We conclude that, with appropriate optimization and validation, the comet assay holds promise as a robust tool for verifying irradiation treatment in quarantine settings, offering an approach for simultaneously assessing both pests and produce.