Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction

Osman, Fatima; Vidalakis, Georgios

doi:10.1007/978-1-0716-1464-8_16

Cited by 2 publications

(4 citation statements)

References 17 publications

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“…Although all three RNA isolation methods were proven valuable and could have different citrus applications, the consistency of the RNA quality isolated from citrus tissues with the semi-automated MagMAX ™ method, as previously reported for grapevine and lily (Osman et al, 2012;Sun et al, 2014) allowed its application in the high-throughput Citrus Nursery Stock Pest Cleanliness Program in California. Beyond the development and validation of the MagMAX ™ -RT-qPCR system, its successful implementation was supported by the integration of robotic pipetting systems and a series of standard operating procedures for swab testing and decontamination protocols allowing the technical personnel, regardless of experience level, to use the method with consistent results with little or no troubleshooting needs (Osman and Vidalakis, 2022;Vidalakis et al, 2022;Dang et al, 2022). This systems approach allowed the use of MagMAX ™ isolated nucleic acids not only for pathogen detection but also for high-throughput sequencing-based microbiome field and greenhouse studies for different citrus tissue types (i.e., stems, leaves, and roots) (Ginnan et al, 2018;Ginnan et al, 2020;Pagliaccia et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…Universal RT-qPCR reactions (Saponari et al, 2008;Vidalakis and Wang, 2013;Chambers et al, 2018;Vidalakis et al, 2022) were performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and multiplex RT-qPCR reactions (Osman et al, 2015;Osman et al, 2017;Osman and Vidalakis, 2022) were carried out using the QuantStudio 12K Flex System (Thermo Fisher Scientific, Waltham, MA).RT-qPCR data was collected and analyzed with the Bio-Rad CFX Manager version 3.1 and the QuantStudio Flex software version 1.3, respectively.…”

Section: Rna Quality Assessment and Rt-qpcr Pathogen Detectionmentioning

confidence: 99%

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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

Dang

Bodaghi²,

Osman³

et al. 2022

Front. Agron.

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Quality Assessment and Rt-qpcr Pathogen Detectionmentioning

confidence: 99%

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

Dang

Bodaghi²,

Osman³

et al. 2022

Front. Agron.

Self Cite

View full text Add to dashboard Cite

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“…More recently, singleplex and multiplex reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and qPCR have drawn more attention and have been extensively utilized in detecting citrus pathogens due to their convenience and elevated levels of sensitivity and specificity [6,7,18,[25][26][27]. They both allow for the simultaneous detection of several pathogens in a single well and give more reliable and reproducible results through quantification compared to conventional PCR and other approaches [26,27]. Although qPCR-based techniques are sensitive, they are still some critical steps away from being practically applied in the field.…”

Section: Introductionmentioning

confidence: 99%

“…Although qPCR-based techniques are sensitive, they are still some critical steps away from being practically applied in the field. Sample preparation is one of the most challenging parts because it requires multiple procedures to extract high-quality nucleic acids from plant tissues [27,28]. Thus, the lack of appropriate sample preparation makes RT-qPCR/qPCR strategies unsuited for in-field diagnosis.…”

Section: Introductionmentioning

confidence: 99%

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Liu,

Bodaghi,

Vidalakis

et al. 2024

Chemosensors

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Effective pathogen detection is essential for plant disease control. However, plant sample preparation for downstream assays, such as quantitative polymerase chain reaction (qPCR), is challenging to perform outside of a laboratory. This paper reports two sample preparation methods featuring chemical and mechanical lysis and nucleic acid extraction using a micro-homogenizer, followed by serial dilution or nucleic acid purification with a paper disk before assay. Five minutes of lysis and extraction resulted in DNA and RNA yields of up to 76.5% and 63.3%, respectively, compared to mortar and pestle controls. Crude lysates were unsuitable for direct use in qPCR assays; however, serial dilution or quick wash using chromatography paper rendered samples ready for such assays. Additionally, the nucleic acids stored on paper disks under various storage conditions remained stable for one month. These methods can facilitate the in-field preparation of citrus samples and allow for both onsite and mail-in diagnostics for growers.

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Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction

Cited by 2 publications

References 17 publications

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

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

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

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