Biology subjects

Harding, M.

Publications and source records attributed to Harding, M..

10 recordsLinked to original sources

bioRxiv2026

A sensitive bioassay for detecting Plasmodiophora brassicae in canola field soils

We describe a biodegradable-cup bioassay for detecting viable Plasmodiophora brassicae in soil samples. Soil samples either artificially inoculated with P. brassicae resting spores or collected from canola fields were aliquoted into biodegradable cups containing 20 g of soil per cup. Two cups representing the same soil sample or inoculum concentration were placed in each pot filled with Sunshine Mix. Six seeds of the canola cultivar Westar were sown into each cup and thinned to four seedlings per cup ten days after planting. After four weeks, roots were examined for the presence of clubroot galls. Across three independent inoculated-soil experiments, galls were observed in samples containing as few as 1 resting spore g-1 soil. In contrast, under a qPCR assay evaluated in parallel, consistent amplification across three technical replicates was obtained only at 100 resting spores g-1 soil or greater. In field samples, the bioassay produced galls from 11 qPCR-positive samples and seven of ten qPCR-negative samples. Although the bioassay is not intended for rapid diagnosis or direct quantification, it provides a practical tool for annual clubroot surveys and for studies requiring recovery, propagation, or characterization of viable P. brassicae from soil samples collected across diverse geographic regions.

microbiology↗

bioRxiv2026

On the sensitivity of qPCR diagnostics for the canola clubroot pathogen Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other Brassica crops. Polymerase chain reaction (PCR), particularly probe-based quantitative PCR (qPCR), is widely used for the detection of P. brassicae in soil samples. To improve consistency in clubroot detection while maintaining efficiency, diagnostic laboratories would benefit from adopting a single, highly efficient qPCR system for routine testing. In this study, we analyzed the primer and probe sequences of all published PCR and qPCR systems for P. brassicae detection. Based on these analyses, three independently developed probe-based qPCR systems were selected and their performance was evaluated using synthesized target DNA (gBlock). One probe-based qPCR system exhibiting superior sensitivity on gBlock was subsequently evaluated on P. brassicae genomic DNA. This system consistently detected DNA equivalent to four resting spores per reaction, corresponding to a soil sample containing 1,000 spores per g soil when the DNA extraction protocol was considered as a component of the qPCR system. The sensitivity of the system was further validated using DNA extracted from soil samples collected from multiple locations across Alberta, where P. brassicae was detected at levels below those associated with visible clubroot symptoms. Based on these results, we recommend this qPCR system for routine clubroot diagnostics in laboratories across Canada.

microbiology↗

bioRxiv2025

Temporal Dynamics of Urinary Extracellular Vesicle Excretion and Cargo in Healthy Subjects over 24 Hours

Urinary extracellular vesicles (uEVs) are promising non-invasive biomarkers of renal physiology and pathology, yet little is known about their temporal excretion patterns or optimal normalization strategies as urine concentrations vary significantly during the day. In this basic physiology study, we used a uniquely designed processing protocol of urine collection to characterize the variation of uEV excretion and cargo throughout a 24 hour period in healthy subjects. Thirteen healthy adults collected every individual void over 24 hours and these voids were independently analyzed along with a composite sample intended to recreate a 24 hour collection sample. uEVs were enriched by low-speed centrifugation with three low-ionic-strength washes to minimize uromodulin contamination. Size, concentration, morphology and cargo of uEVs were characterized with nanoparticle tracking analysis, cryo-electron microscopy, immunoblotting and single EV flow cytometry. We observed a stable urine creatinine excretion into the urine over the day. uEV concentrations varied up to 100-fold across the day between individuals with a decreasing trend over time with the result close to reaching statistical significance (p=0.06). uEV concentration was positively associated with urine creatinine, specific gravity, and TSG101, an EV marker. The association between urine flow and uEV excretion varied across the day. Most tested uEV cargo markers exhibited substantial within individual variation over the day without consistent trends in our healthy cohort. When grouped into predefined time periods to visualize temporal patterns, a transient dip at noon was observed, followed by a significant increase in the subsequent period, consistent with a pattern previously reported. These findings offer foundational insights into uEV excretion patterns over the course of a day in a healthy cohort and emphasize key methodological considerations essential for advancing uEV biomarker research.

physiology↗

bioRxiv2025

RNAse-free manufacture of Venezuelan Equine Encephalitis Virus (VEEV) plasmid DNA vaccine

The long shelf-life and stability of DNA makes this platform highly attractive for low-cost, rapid delivery of pandemic response vaccines. Protocols utilized for clinical grade plasmid manufacture by contract development and manufacturing organizations are not readily accessible to academic and public research laboratories engaged in early-phase plasmid vaccine development. We present here the framework for DNA manufacturing using 3L-scale fermentation, anion-exchange chromatography and tangential flow filtration (TFF) leading to RNAase-free manufacture of plasmid DNA. The Venezuelan Equine Encephalitis Virus vaccine plasmid pWRG/VEEV, encoding the glycoprotein (E)3, E2, 6K and E1 genes was used as the prototype for this process development. The current effort yielded >95% pure and >80% supercoiled pWRG/VEEV plasmid preparations at 50-g wet cell weight scale. These data showed feasibility of manufacturing, highly pure pWRG/VEEV plasmid DNA using a cGMP compliant manufacturing process. Distribution StatementApproved for public release: distribution is unlimited

biochemistry↗

bioRxiv2024

Development of a qPCR assay and a LAMP assay for Verticillium longisporum detection and a triplex qPCR assay for simultaneous detection of V. longisporum, Leptosphaeria biglobosa and L. maculans from canola samples

Verticillium wilt, Verticillium stem striping, and Verticillium stripe, are common disease names that all denote infection caused by Verticillium longisporum, on canola, or other Brassica crops. In this study, a quantitative PCR (qPCR) assay and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-{micro} L reaction or 1,400 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg.

microbiology↗

bioRxiv2023

Detection and differentiation of Xanthomonas translucens pv. translucens and pv. undulosa from wheat and barley by duplex quantitative PCR

Two probe-based quantitative PCR (qPCR) systems, namely P-Xtt and P-Xtu, were developed for diagnosis of cereal bacterial leaf streak pathogens Xanthomonas translucens pv. translucens and pv. undulosa, respectively. P-Xtt is specific to pv. translucens. P-Xtu is specific to pv. undulosa, pv. cerealis, pv. secalis and pv. pistaciae. P-Xtt and P-Xtu worked on all accessible strains of pv. translucens and pv. undulosa, respectively. Both systems could detect 100 copies of the target gBlock DNA. The two systems could be used in both singleplex qPCR and duplex qPCR with similar efficiencies. On genomic DNA from strains of various X. translucens pathovars, both singleplex qPCR and duplex qPCR could specifically detect and differentiate pv. translucens and pv. undulosa. The duplex qPCR could detect pv. translucens and pv. undulosa from genomic DNA of 1,000 bacterial cells. On infected barley and wheat grain samples, and on one infected wheat leaf sample, the duplex qPCR showed similar efficiency compared to a previously published qPCR system but with the additional capability of pathovar differentiation. The duplex qPCR system developed in this study will be useful in studies on bacterial leaf streak and detection/differentiation of the pathogens.

plant biology↗

bioRxiv2021

Detection of Xanthomonas translucens pv. undulosa from wheat by quantitative PCR

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pv. undulosa (Xtu). The protocol can also detect X. translucens pv. translucens (Xtt), but cant differentiate the two pathovars. When tested on DNA from plant, non-target bacteria and culture of microorganisms from wheat seeds, the qPCR showed a high specificity. On purified Xtu DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of Xtu cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing [≥]1,000 cells. Since 2 {micro}L of the total of 50 {micro}L DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected seed and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one seed. Thus, the qPCR system could theoretically detect Xtu and/or Xtt in samples where the bacteria had an average concentration [≥]40 cells per seed.

microbiology↗

bioRxiv2021

Plasmodiophora brassicae infection threshold - How many resting spores are required for infection of canola (Brassica napus)

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as [≤] 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.

microbiology↗

bioRxiv2020

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV)

Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65{degrees}C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used rt-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

microbiology↗

bioRxiv2019

Plasmodiophora brassicae in its environment-effects of temperature and light on resting spore survival in soil

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot has been challenging, due largely to the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 x 107 spores g-1 soil) were stored at various temperatures for two years. Additionally, other samples were buried in soil, or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative polymerase chain reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4{degrees}C, 20{degrees}C and being buried in the soil were better conditions for spore survival than were -20{degrees}C, 30{degrees}C and at the soil surface. Most of the spores kept on the soil surface were killed, suggesting the negative effect of light on spore viability. Additional experiments confirmed that ultraviolet (UV) light contributed a large negative effect on spore viability as lower pathogenicity and less DNA content were observed from the 2-and 3-hour UV light treated spores compared to the untreated control. Finally, this work demonstrated that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential due to the presence and persistence of DNA from dead spores.

microbiology↗