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

Area of Science:

• Molecular biology
• Plant pathology
• Virology

Background:

• Accurate detection of plant viruses and viroids is crucial for disease management.
• Existing methods can be time-consuming, costly, or require extensive sample preparation.
• Simultaneous detection of multiple pathogens simplifies diagnostics.

Purpose of the Study:

• To present a protocol for dual interrogation RT-qPCR for simultaneous viroid and virus genotyping in plants.
• To enable rapid, high-throughput, and economical molecular diagnostics.
• To facilitate field-deployable testing using crude nucleic acid extracts.

Main Methods:

• Utilized dual interrogation molecular assays for simultaneous detection.
• Employed inhibitor-tolerant polymerases for robustness with crude extracts.
• Developed a protocol for nucleic acid extraction from raw tissue or FTA cards.
• Applied probe-based RT-qPCR for genotyping.

Main Results:

• Demonstrated a rapid and high-throughput method for pathogen detection.
• Achieved simultaneous detection of viroid and virus genotypes.
• Showcased the utility of crude nucleic acid extracts, including from FTA cards.
• Ensured high accuracy and robustness with challenging sample types.

Conclusions:

• The dual interrogation RT-qPCR protocol offers an efficient and reliable approach for plant pathogen screening.
• The method streamlines sample preparation, enabling field-deployable diagnostics.
• This protocol supports effective plant disease management through accurate genotyping.