ABSTRACT
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.
Subject(s)
Citrus , Viroids , Citrus/genetics , Nucleic Acids , RNA , RNA, Viral/genetics , Viroids/geneticsABSTRACT
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.
Subject(s)
Citrus , Viroids , Plant Diseases , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Viroids/geneticsABSTRACT
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.
Subject(s)
Citrus , Viroids , Citrus/genetics , Plant Diseases , Viroids/geneticsABSTRACT
Citrus tristeza virus (CTV) isolates collected from citrus germplasm, dooryard and field trees in California from 1914 have been maintained in planta under quarantine in the Citrus Clonal Protection Program (CCPP), Riverside, California. This collection, therefore, represents populations of CTV isolates obtained over time and space in California. To determine CTV genetic diversity in this context, genotypes of CTV isolates from the CCPP collection were characterized using multiple molecular markers (MMM). Genotypes T30, VT, and T36 were found at high frequencies with T30 and T30+VT genotypes being the most abundant. The MMM analysis did not identify T3 and B165/T68 genotypes; however, biological and phylogenetic analysis suggested some relationships of CCPP CTV isolates with these two genotypes. Phylogenetic analysis of the CTV coat protein (CP) gene sequences classified the tested isolates into seven distinct clades. Five clades were in association with the standard CTV genotypes T30, T36, T3, VT, and B165/T68. The remaining two identified clades were not related to any standard CTV genotypes. Spatiotemporal analysis indicated a trend of reduced genotype and phylogenetic diversity as well as virulence from southern California (SC) at early (1907-1957) in comparison to that of central California (CC) isolates collected from later (1957-2009) time periods. CTV biological characterization also indicated a reduced number and less virulent stem pitting (SP) CTV isolates compared to seedling yellows isolates introduced to California. This data provides a historical insight of the introduction, movement, and genetic diversity of CTV in California and provides genetic and biological information useful for CTV quarantine, eradication, and disease management strategies such as CTV-SP cross protection.