Ratti Claudio, Minguzzi Stefano, Turina Massimo
Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Bologna, Italy.
Institute for Sustainable Plant Protection, National Research Council of Italy, IPSP-CNR, Turin, Italy.
Methods Mol Biol. 2019;1875:159-169. doi: 10.1007/978-1-4939-8837-2_13.
Most of the molecular diagnostic protocols used for phytoplasmas detection are based on the purification of total nucleic acids and on the use of genomic DNA of the pathogen as the target of amplification. Here we describe a diagnostic approach that, avoiding the purification of nucleic acids and exploiting the amplification of the abundant phytoplasma ribosomal RNA molecules produced during the infectious process, allows reducing the time and the costs necessary for the analysis, without affecting sensitivity and specificity. This is useful in particular when high numbers of analyses are required, as in certification programs, to monitor phytoplasmas classified as quarantine or quality pathogens. The protocol here described can be used for the detection and quantification of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani by qPCR, RT-qPCR, ddPCR, and ddRT-PCR techniques based on TaqMan chemistry.
用于植原体检测的大多数分子诊断方案都基于总核酸的纯化，并以病原体的基因组DNA作为扩增靶点。在此，我们描述了一种诊断方法，该方法无需纯化核酸，而是利用感染过程中产生的丰富的植原体核糖体RNA分子进行扩增，从而在不影响灵敏度和特异性的情况下，减少分析所需的时间和成本。这在需要大量分析的情况下特别有用，例如在认证计划中，用于监测被归类为检疫或质量病原体的植原体。本文所述方案可用于通过基于TaqMan化学的qPCR、RT-qPCR、ddPCR和ddRT-PCR技术检测和定量苹果植原体、梨植原体、李植原体、葡萄植原体和茄植原体。
