Detection and identification of transgenic virus resistant papaya and squash by multiplex PCR

Two separate multiplex PCR assays were developed for the detection of transgenic papaya and transgenic squash. Papaya line 55-1 contains a genetic insertion consisting of the coat protein gene of Papaya ringspot virus strain p and is resistant to infection by this virus. A multiplex PCR was developed to specifically amplify the papaya ringspot virus coat protein transgene construct and an endogenous papaya gene sequence. A third primer set was designed to amplify the β-glucuronidase gene construct, which can distinguish between the commercial and noncommercial papaya lines 55-1 and 63-1. Squash line ZW-20 contains genetic insertions of the coat protein genes from Watermelon mosaic virus II and Zucchini yellow mosaic virus and is resistant to these two viruses. Squash line CZW-3 is similar to ZW-20 but additionally confers resistance to Cucumber mosaic virus. A second multiplex assay was developed to detect and differentiate squash lines ZW-20 and CZW-3 in a single reaction.