A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

Abstract

Citrus germplasm programs can benefit from high-throughput polymerase chain reaction (PCR)-based methods for the detection of graft-transmissible pathogens in propagative materials. These methods increase diagnostic capacity, and thus contribute to the prevention of disease spread from nurseries to citrus orchards. High quality nucleic acids, as determined by purity, concentration, and integrity, are a prerequisite for reliable PCR detection of citrus pathogens. Citrus tissues contain high levels of polyphenols and polysaccharides, which can affect nucleic acid quality and inhibit PCR reactions. Various commercially available RNA isolation methods are used for citrus and include: phenol-chloroform (TRIzol®, Thermo Fisher Scientific); silica columns (RNeasy® Plant Mini Kit, Qiagen); and magnetic beads-based methods (MagMAX™-96 Viral RNA Isolation Kit, Thermo Fisher Scientific). To determine the quality of RNA and its impact on the detection of graft-transmissible citrus pathogens in reverse transcription (RT) PCR-based assays, we compared these three RNA isolation methods. We assessed RNA purity, concentration, and integrity from citrus inoculated with different viruses and viroids. All three RNA isolation methods produced high quality RNA, and its use in different RT-PCR assays resulted in the detection of all targeted citrus viruses and viroids with no false positive or negative results. TRIzol® yielded RNA with the highest concentration and integrity values but some samples required serial dilutions to remove PCR inhibitors and detect the targeted pathogens. The RNeasy® kit produced the second highest concentration and purity of RNA, and similar integrity to TRIzol®. MagMAX™ isolation also provided high quality RNA but most importantly produced RNA with consistent results clustered around a median value for concentration, purity, and integrity. Subsequently, MagMAX™-96 was combined with the semi-automated MagMAX™ Express-96 Deep Well Magnetic Particle Processor, for high-throughput sample processing. MagMAX™-96 enabled the diagnostic laboratory of the Citrus Clonal Protection Program-National Clean Plant Network at the University of California, Riverside to process over 16,500 samples from citrus budwood source trees between 2010 and 2019. This high-throughput approach dramatically reduced the incidence of viroids in citrus nurseries and was key to the successful implementation of the mandatory Citrus Nursery Stock Pest Cleanliness Program in California.

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples

“…Our data demonstrates that the sensitivity of DWV detection by both RT-qPCR and nanopore sequencing is comparable after total RNA extraction by NucleoSpin® Virus and NucleoMag™ Virus isolation kits. Both methods provided similar analytical results obtained from RT-qPCR showing that both DWV strain loads and C t values from honey bee pooled samples processed by affinity purification columns and magnetic beads-based technology method correlated well, which is in agreement with previous work [ 37 , 38 ]. Differences observed in the strength of amplification signal could be due to higher RNA yields and purity delivered by NucleoSpin®.…”

“…To perform virus screening with qPCR, nucleic acid (RNA or DNA) extraction is often the first step in the screening process. Nucleic acid isolation is initiated with mechanical or chemical disruption of cells, following a purification step by precipitation with ethanol/isopropanol, affinity purification columns or magnetic beads-based technology [ 33 – 38 , 42 , 43 ]. A common approach in bee research is based on using manual methods of extraction utilizing organic solvents or affinity purification columns kits, or a combination of both.…”

“…PCR remains one of the most favored and applied techniques for virus detection and disease diagnosis. It is routinely used for virus diagnostics in many research fields and is a standard for evaluating viral loads in biomedical research, agricultural and environmental sectors [33][34][35][36][37][38]. NGS technology is an additive and sometimes alternative method, capable of identifying unknown viruses in the sample, including variants and quasispecies [39][40][41], and is a reliable tool for validation of PCR results [32,39,40,41].…”