Torres Anthony, Gaudino Reginald
Cannabis Research Institute, Discovery Partners Institute, University of Illinois System, Chicago, IL, USA.
Front Range Biosciences, Lafayette, CO, USA.
Methods Mol Biol. 2025;2943:117-130. doi: 10.1007/978-1-0716-4642-7_10.
This chapter presents a protocol for dual interrogation reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) genotyping, a rapid, high-throughput, and economical molecular diagnostic approach for the simultaneous detection of viroid and virus genotypes in plant samples. The method utilizes crude nucleic acid extracts from raw tissue or FTA cards, streamlining sample preparation and enabling field-deployable testing. The incorporation of dual interrogation molecular assays and inhibitor-tolerant polymerases ensures high accuracy and robustness, even with challenging sample types. The protocol details the extraction process and subsequent RT-qPCR detection, facilitating efficient and reliable pathogen screening for plant diagnostics and disease management. In this workflow, we describe an extraction and probe-based RT-qPCR method used to detect viroid and virus genotypes in plant samples extracted from raw tissue or FTA imprinted cards.
本章介绍了一种用于双重检测逆转录定量聚合酶链反应（RT-qPCR）基因分型的方案，这是一种快速、高通量且经济的分子诊断方法，用于同时检测植物样品中的类病毒和病毒基因型。该方法利用来自原始组织或FTA卡的粗核酸提取物，简化了样品制备过程，并实现了可现场部署的检测。双重检测分子测定法和耐抑制剂聚合酶的结合确保了即使对于具有挑战性的样品类型也具有高精度和稳健性。该方案详细介绍了提取过程及后续的RT-qPCR检测，有助于为植物诊断和病害管理进行高效且可靠的病原体筛查。在本工作流程中，我们描述了一种基于提取和探针的RT-qPCR方法，用于检测从原始组织或FTA印记卡中提取的植物样品中的类病毒和病毒基因型。
