Probe-Based RT-qPCR Workflow for Dual Interrogation of Virus/Viroid Genotype in Molecular Diagnostics Multiplex.

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.