automating-citrus-budwood-processing-for-downstream-pathogen

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

University of California Riverside

High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.

High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids.

Quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR have now become the gold standard for molecular diagnostics because of its sensitivity, specificity, and reproducibility. In addition, qPCR diagnostics are flexible because they can be scaled for high- or low-throughput applications. Here we describe an optimized assay and workflow for the universal detection of eight citrus viroid species and their variants by RT-qPCR. The assay allows for quick and efficient molecular detection of viroids without the need to run RT-qPCR for each individual viroid species.

SYBR Green RT-qPCR for the Universal Detection of Citrus Viroids.

The QuantiGene Plex assay is a molecular non-polymerase chain reaction (PCR)-based multiplex method adapted for citrus viroid detection and identification. Here, we describe the procedures to utilize the QuantiGene Plex assay as a high-throughput screening tool for viroids in purified or crude RNA extracts from citrus tissues.

Article	Year
High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34845684	2022
SYBR Green RT-qPCR for the Universal Detection of Citrus Viroids. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34845697	2022
QuantiGene Plex Assay: A Method for High-Throughput Multiplex Citrus Viroid Detection and Identification. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34845700	2022

Irene Lavagi-Craddock

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