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

摘要

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~R 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~R) or in a quantitative real-time assay (XRT), and Discovery kits applicable to any pathogen. In either assay ofAcceler8~R 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~R Acceler8~R Kit for PPV in 2014, and has also developed the AmplifyRP~R 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~R assay, AmplifyRP~R XRT* (formerly known as Combo AmplifyRP), in which both XRT and Acceler8~R assays were combined and performed as one reaction in a single PCR tube. As a result, both quantitative realtime fluorescence data and qualitative endpoint product visual results were rapidly achieved through a single reaction in a single tube. With this AmplifyRP~R XRT+ method, we were able to detect all strains of PPV in crude plant extracts or purified RNA, as do Agdia's AmplifyRP~R Acceler8~R and XRT kits. AmplifyRP~R XRT+ can be performed with a portable fluorescence reader, a real-time PCR machine and lateral flow strip. The sensitivity of AmplifyRP~R XRT+ was comparable to those of our existing AmplifyRP~R Acceler8~R and XRT assays. In addition, AmplifyRP~R XRT+ preserves the simplicity and rapidity of bothAmplifyRP~R Acceler8~R and XRT, and opens up a great opportunity for rapid high-throughput screening for PPV and other plant pathogens through isothermal amplification.