Development of a novel isothermal AmplifyRP® assay for rapid detection of Plum pox virus - a real-time and endpoint assay in a single PCR tube
Recombinase polymerase amplification, a leading isothermal amplification technology, has been increasingly used in detection of nucleic acids. Utilizing this technology, Agdia Inc. developed an AmplifyRP® platform for rapid detection of plant pathogens such as Plum pox virus (PPV). Currently available are pathogen-specific AmplifyRP® tests, either in a qualitative endpoint assay (Acceler8®) or in a quantitative real-time assay (XRT), and Discovery kits applicable to any pathogen. In either assay of Acceler8® or XRT, two target-specific primers and one internal probe are used, and all specific recombination and amplification occurs rapidly at a single constant temperature of 39°C. Agdia commercialized the AmplifyRP® Acceler8® Kit for PPV in 2014, and has also developed the AmplifyRP® XRT assay for PPV in addition to Agdia's existing PPV ELISA and ImmunoStrip kits. In this study, we report the development of a novel isothermal AmplifyRP® assay, AmplifyRP® XRT+ (formerly known as Combo AmplifyRP), in which both XRT and Acceler8® assays were combined and performed as one reaction in a single PCR tube. As a result, both quantitative real-time fluorescence data and qualitative endpoint product visual results were rapidly achieved through a single reaction in a single tube. With this AmplifyRP® XRT+ method, we were able to detect all strains of PPV in crude plant extracts or purified RNA, as do Agdia's AmplifyRP® Acceler8® and XRT kits. AmplifyRP® XRT+ can be performed with a portable fluorescence reader, a real-time PCR machine and lateral flow strip. The sensitivity of AmplifyRP® XRT+ was comparable to those of our existing AmplifyRP® Acceler8® and XRT assays. In addition, AmplifyRP® XRT+ preserves the simplicity and rapidity of both AmplifyRP® Acceler8® and XRT, and opens up a great opportunity for rapid high-throughput screening for PPV and other plant pathogens through isothermal amplification.
Zhang, S., Russell, P., McOwen, N., Davenport, B. and Li, R. 2017. Development of a novel isothermal AmplifyRP® assay for rapid detection of Plum pox virus - a real-time and endpoint assay in a single PCR tube. Acta Hort. (ISHS) 1163:31-38
AmplifyRP® XRT+, isothermal amplification, lateral flow assay, Plum pox virus, real-time assay, recombinase polymerase amplification