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.

在植物病毒诊断领域，可靠的多目标检测方法实施是主要且最具挑战性的目标之一。本研究中，我们报告了一种高度稳健的多重PCR（HiPlex）优化版本的开发及其有效性，该版本能够同时对14种苹果梨树病毒和5种苹果梨树航标进行靶向扩增子测序。利用我们先前开发的HiPlex对数百个诊断样本的扩增子测序数据分析，使我们能够识别出针对苹果梨树病毒和航标的每种靶向病毒和航标的最高效和最特异的引物对，同时采用了新设计的引物。这导致从新的组装中选出了116对引物组成新的HiPlex（命名为HiPlex v.2），分别减少了引物和多重PCR反应的数量60%和50%。除了原始HiPlex中包含的17种苹果梨树病毒和航标外，此第二个版本还包括了两种内波病毒、番茄环斑病毒和烟草环斑病毒。优化后的引物集通过减少背景扩增，提高了方法的成本效益和灵敏度。即使在10,000倍稀释的情况下，使用HiPlex v.2和本研究中包含的所有病毒和航标的单个RT-qPCRs检测病毒和航标的结果也具有可比性。这一新的HiPlex代表了针对苹果梨树病毒和航标大规模筛查的一种强大、灵敏且成本效益高的替代方案。