QUANTITATIVE ASSESSMENT OF METHODS FOR BACTERIAL AND VIRAL PURIFICATION AND CONCENTRATIONAbstract
The COVID-19 global pandemic has led to the exploration and implementation of rapid tests for viral load identification and thus, control of the spread. For this, there are varying approaches with the gold standard being quantitative Polymerase Chain Reaction and a potential more rapid alternate approach being the use of an antibody coated biosensor such as a field effect transistor (FET). Analogously, bacterial pathogens such as anthrax need sensing and quantification as well. Therefore, methods for pathogen collection and detection for both viruses and bacteria are needed.
Regardless of the analysis technique, it is ideal for samples to contain little to zero non-target particles or contaminants that might interfere with the detection approach. As such, traditional ways of purifying and concentrating samples prior to any type of analysis involve both chemical, physical, physiochemical, or biological approaches such as filtration, centrifugation, affinity chromatography, immunomagnetic separation, etc. Of these, we will focus on the physical approach, filtration, due to its simplicity, low cost, varying options, and ability to process large and small sample volumes. In this work, we investigate how well the method works for virus or bacteria in the presence of high interferent concentrations that could potentially be present in saliva samples or other hydrosol samples taken from an environment to be tested. The research presented here characterizes the applicability of syringe filters and a tangential flow filtration device for the purification and concentration of bacteria and virus samples, respectively. Furthermore, automation of such systems was explored.
Varying syringe filter pore sizes yielded different recoveries of bacteria for purifying and concentrating the sample. Furthermore, varying volumes were analyzed for xviii optimal recovery and concentration of the target. We also developed a fully automated method for double filter filtration to enable hands-free purification and concentration in 5.5 minutes for 5 mL of input volume with a 42 ± 13-fold enrichment improvement (n = 3). Furthermore, the purification and concentration of virus using a manually operated tangential flow filtration device was also explored and yielded modest concentration increases of around 2× with an enrichment improvement of up to 1,916 ± 1,839-fold (n = 3) under one configuration. By characterizing and automating these readily available items, we can enhance the detection of samples by decreasing labor time and processing complexity required for the purification and concentration of the target pathogens.
Improving Soil Quality and Potato Productivity with Manure and High-Residue Cover Crops in Eastern CanadaAbstract
Under intensive low residue agricultural systems, such as those involving potato (Solanum tuberosum L.)-based systems, stagnant crop yields and declining soil health and environmental quality are common issues. This study evaluated the effects of pen-pack cow (Bos Taurus) manure application (20 Mg·ha−1) and cover crops on nitrate dynamics and soil N supply capacity, subsequent potato yield, selected soil properties, and soil-borne disease. Eight cover crops were tested and included grasses, legumes, or a mixture of legumes and grasses, with red clover (Trifolium pratense L.) used as a control. Forage pearl millet (Pennisetum glaucum L.) was associated with highest dry matter. On average, red clover had 88% higher total N accumulation than the treatments mixing grasses and legumes, and the former was associated with higher soil nitrate in fall before residue incorporation and overwinter, but this was not translated into increased potato yields. Pearl millet and sorghum sudangrass (Sorghum bicolor × sorghum bicolor var. Sudanese) were associated with lower soil nitrate in comparison to red clover while being associated with higher total potato yield and lower numerical value of root-lesion nematodes (Pratylenchus penetrans), although this was not statistically significant at 5% probability level. Manure incorporation increased total and marketable yield by 28% and 26%, respectively, and increased soil N supply capacity by an average of 44%. Carbon dioxide released after a short incubation as a proxy of soil microbial respiration increased by an average of 27% with manure application. Our study quantified the positive effect of manure application and high-residue cover crops on soil quality and potato yield for the province of Prince Edward Island.
Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR testAbstract
The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum, phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi-selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water.
Sensitive, quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite speciesAbstract
Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms’ epidemiology and pathogenesis.
The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.
Phenotypic characterization of genetically distinct Phytophthora cinnamomi isolates from avocadoAbstract
Phytophthora cinnamomi, the causal agent of Phytophthora Root Rot (PRR), is the most destructive disease of avocado worldwide. A previous study identified two genetically distinct clades of A2 mating type avocado isolates in California, however the phenotypic variation among them was not assessed. This study described the phenotype of a subset of isolates from these groups regarding growth rate, growth temperature, virulence, and fungicide sensitivity. Isolates corresponding to the A2 clade I group exhibited higher mycelial growth rate and sensitivity to higher temperatures than other isolates. Among the fungicides tested, potassium phosphite had the highest 50% effective concentration for mycelial growth inhibition and oxathiapiprolin had the lowest. Mycelial growth rate and potassium phosphite sensitivity phenotypes correlate with specific group of isolates suggesting that these traits could be a group characteristic. Moreover, isolates that are more virulent in avocado and less sensitive to potassium phosphite were identified. A detached leaf P. cinnamomi inoculation method using Nicotiana benthamiana was developed and validated providing an alternative method for assessing the virulence of a large number of isolates. This information will help avocado PRR management and assist breeding programs for the selection of rootstocks resistant against a more diverse pathogen population.
Development of Molecular Methods to Detect Macrophomina phaseolina from Strawberry Plants and SoilAbstract
Macrophomina phaseolina is a broad-host range fungus that shows some degree of host preference on strawberry, and causes symptoms including crown rot and root rot. Recently, this pathogen has impacted strawberry production as fumigation practices have changed, leaving many growers in California and around the world in need of accurate, rapid diagnostic tools for M. phaseolina in soil and infected plants. This study uses next-generation sequencing and comparative genomics to identify a locus that is unique to isolates within a main genotype shared by a majority of isolates that infect strawberry. This locus was used to develop a quantitative single-tube nested TaqMan qPCR assay which is able to quantify as little as 2-3 microsclerotia/g of soil with 100% genotype specificity. An isothermal assay using recombinase polymerase amplification (RPA) was developed from the same locus and has been validated on over 200 infected strawberry plants with a diagnostic sensitivity of 93% and a diagnostic specificity of 99%, respectively. Together, this work demonstrates the value of using new approaches to identify loci for detection and provides valuable diagnostic tools that can be used to monitor soil and strawberry plant samples for M. phaseolina.
Assessment of Erysiphe necator ascospore release models for use in the Mediterranean climate of Western OregonAbstract
Predictive models have been developed in several major grape-growing regions to correlate environmental conditions to Erysiphe necator ascospore release; however, these models may not be broadly applicable in regions with different climatic conditions. To assess ascospore release in near-coastal regions of western Oregon, chasmothecia (syn. cleistothecia) were collected prior to leaf drop and placed onto natural and artificial grape trunk segments and overwintered outside. Ascospore release was monitored for three overwintering seasons using custom impaction spore traps from leaf drop (BBCH 97) until the onset of the disease epidemic in the following growing season. Airborne inoculum was concurrently monitored in a naturally infested research vineyard. Weather and ascospore release data were used to assess previously-developed models, and correlate environmental conditions to ascospore release. Ascospore release was predicted by all models prior to bud break (BBCH 08), and was observed from the first rain event following the start of inoculum monitoring until monitoring ceased. Previously developed models over-predicted ascospore release in the Willamette Valley and predicted exhaustion of inoculum prior to bud break. The magnitude of ascospore release could not be correlated to environmental conditions, thus a binary ascospore release model was developed where release is a function of the collective occurrence of the following factors within a 24-hour period: > 6 hours of cumulative leaf wetness during temperatures > 4 °C, precipitation > 2.5 mm, and relative humidity > 80%. The Oregon Model was validated using field collected ascospore datasets, and predicted ascospore release with 66% accuracy (P = 0.02). Extant methods for estimating ascospore release may not be sufficiently accurate to use as predictive models in wet, temperate climatic regions.
Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditionsAbstract
Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in NepalAbstract
This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.
Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancerAbstract
Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results.
Detection and Quantification of Acute Myeloid Leukemia-Associated Fusion TranscriptsAbstract
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.