Development of Qualitative Real-time PCR Assays for Detecting Genetically Modified Squash Events ZW20 and CZW3

In March 2023, unapproved genetically modified (GM) squashes were discovered in South Korea. These were thought to be GM squash ZW20 and CZW3, which are approved for use as food only in Canada and the United States. Because the safety of ZW20 and CZW3 has not been evaluated in Japan, they must continue to be monitored to prevent their unintentional import. In this study, we developed and validated real-time PCR-based qualitative detection methods for ZW20 and CZW3. We evaluated these two detection methods on the basis of PCR amplification efficiency, limit of detection, specificity, and reproducibility to determine their utility for distinguishing and identifying ZW20 and CZW3. One method detects the zucchini yellow mosaic virus resistance gene (ZYMV-coat protein: ZYMV-cp) sequence present in both ZW20 and CZW3, whereas the other method detects the cucumber mosaic virus resistance gene (CMV-coat protein: CMV-cp) sequence present only in CZW3. Our results showed that the ZYMV-cp and CMV-cp detection methods are highly specific for GM squash ZW20 and CZW3, and are sufficiently sensitive, capable of detecting transgenes with at least 6.3 and 3.1 copies per reaction, respectively. Based on this study, we developed the official detection method for GM squash in Japan by combining the ZYMV-cp and CMV-cp detection methods to discriminate between ZW20 and CZW3, making it useful for monitoring food safety.