Improved detection of pome viruses and viroids through amplicon-based sequencing for high-throughput diagnostics

The implementation of reliable methods for the detection of multiple targets at once is one of the main and most challenging goals in plant viral diagnosis. In the present study we report the development and effectiveness of an optimized version of a highly robust multiplexed PCR (HiPlex) for simultaneous targeted amplicon sequencing of 14 viruses and five viroids of pome fruit trees. Amplicon sequencing data analyses of hundreds of diagnosed samples using our previously developed HiPlex, enabled the identification of the most efficient and specific primer pairs for each targeted virus and viroid of pome fruit trees while also implementing newly designed primers. This resulted in the selection of 116 primer pairs into a newly assembled HiPlex (dubbed HiPlex v.2), reducing the number of primers and multiplex PCR reactions needed by 60% and 50% respectively. In addition to the 17 pome fruit tree viruses and viroids included in the original HiPlex, this second version also includes two nepoviruses, tomato ring spot virus and tobacco ringspot virus. The optimized set of primers increased the cost-effective of the method as well as the sensitivity by reducing background amplification. Comparable results for viruses and viroids detection were observed using HiPlex v.2 and individual RT-qPCRs for all viruses and viroids included in this study, even at a 10,000-fold dilution. This new HiPlex represents a powerful, sensitive, and high-cost effective alternative for large scale screening of pome fruit trees viruses and viroids.