scholarly journals Detection of blueberry stunt phytoplasma in Eastern Canada using cpn60-based molecular diagnostic assays

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Christine Hammond ◽  
Edel Pérez-López ◽  
Jennifer Town ◽  
Charles Vincent ◽  
Debra Moreau ◽  
...  
Keyword(s):  
Production Management ◽  
Low Cost ◽  
Lamp Assay ◽  
Vaccinium Corymbosum ◽  
Molecular Diagnostic ◽  
Eastern Canada ◽  
Diagnostic Assays ◽  
Growing Seasons ◽  
Pathogen Load ◽  
Interactive Map

AbstractBlueberry stunt phytoplasma (BBSP; ‘Candidatus Phytoplasma asteris’) is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016–2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

scholarly journals Molecular Detection of Blueberry Stunt Phytoplasma in Eastern Canada: a Multi-year Study

2021 ◽  
Author(s):  
Christine Hammond ◽  
Edel Perez-Lopez ◽  
Jennifer Town ◽  
Charles Vincent ◽  
Debra Moreau ◽  
...  
Keyword(s):  
Production Management ◽  
Low Cost ◽  
Lamp Assay ◽  
Vaccinium Corymbosum ◽  
Eastern Canada ◽  
Diagnostic Assays ◽  
Proactive Approach ◽  
Growing Seasons ◽  
Pathogen Load ◽  
Interactive Map

Abstract Blueberry stunt phytoplasma (BBSP; ‘Candidatus Phytoplasma asteris’) is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016–2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in a given plant within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

scholarly journals A Robust, Low-Cost Instrument for Real-time Colorimetric Isothermal Nucleic Acid Amplification

2021 ◽  
Author(s):  
Frank Myers ◽  
Brian Moffatt ◽  
Ragheb El Khaja ◽  
Titash Chatterjee ◽  
Gurmeet Marwaha ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
Diagnostic Assays ◽  
Isothermal Nucleic Acid Amplification ◽  
Point Of Care Diagnostics ◽  
Time Readout

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati

Parasitology ◽  
2021 ◽  
pp. 1-8
Author(s):  
Héctor Gabriel Avila ◽  
Marikena Guadalupe Risso ◽  
Paula Ruybal ◽  
Silvia Analía Repetto ◽  
Marcos Javier Butti ◽  
...  
Keyword(s):  
Low Cost ◽  
Lamp Assay ◽  
Toxocara Canis ◽  
Toxocara Cati

Abstract

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals Genotype-Dependent Tipburn Severity During Lettuce Hydroponic Culture Is Associated with Altered Nutrient Leaf Content

Agronomy ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 616
Author(s):  
Virginia Birlanga ◽  
José Ramón Acosta-Motos ◽  
José Manuel Pérez-Pérez
Keyword(s):  
Low Cost ◽  
Nutrient Content ◽  
Leafy Vegetables ◽  
Production Methods ◽  
Growing Seasons ◽  
Lactuca Sativa L ◽  
Hydroponic Cultivation ◽  
Highly Correlated ◽  
Nutrient Solutions ◽  
Nutrient Traits

Cultivated lettuce (Lactuca sativa L.) is one of the most important leafy vegetables in the world, and most of the production is concentrated in the Mediterranean Basin. Hydroponics has been successfully utilized for lettuce cultivation, which could contribute to the diversification of production methods and the reduction of water consumption and excessive fertilization. We devised a low-cost procedure for closed hydroponic cultivation and easy phenotyping of root and shoot attributes of lettuce. We studied 12 lettuce genotypes of the crisphead and oak-leaf subtypes, which differed on their tipburn resistance, for three growing seasons (Fall, Winter, and Spring). We found interesting genotype × environment (G × E) interactions for some of the studied traits during early growth. By analyzing tipburn incidence and leaf nutrient content, we were able to identify a number of nutrient traits that were highly correlated with cultivar- and genotype-dependent tipburn. Our experimental setup will allow evaluating different lettuce genotypes in defined nutrient solutions to select for tipburn-tolerant and highly productive genotypes that are suitable for hydroponics.

scholarly journals Stories, journeys and smart maps: an approach to universal access

2021 ◽  
Author(s):  
Xi Wang ◽  
Danny Crookes ◽  
Sue-Ann Harding ◽  
David Johnston
Keyword(s):  
Universal Access ◽  
Low Cost ◽  
Research Question ◽  
Emotional Engagement ◽  
New Approach ◽  
Reception Study ◽  
Audio Description ◽  
Museum Experience ◽  
Interactive Map ◽  
Partially Sighted

AbstractThis paper proposes a new approach to universal access based on the premise that humans have the universal capacity to engage emotionally with a story, whatever their ability. Our approach is to present the “story” of museum resources and knowledge as a journey, and then represent this journey physically as a smart map. The key research question is to assess the extent to which our “story” to journey to smart map’ (SJSM) approach provides emotional engagement as part of the museum experience. This approach is applied through the creation of a smart map for blind and partially sighted (BPS) visitors. Made in partnership with Titanic Belfast, a world-leading tourist attraction, the interactive map tells the story of Titanic’s maiden voyage. The smart map uses low-cost technologies such as laser-cut map features and software-controlled multi-function buttons for the audio description (AD). The AD is enhanced with background effects, dramatized personal stories and the ship’s last messages. The results of a reception study show that the approach enabled BPS participants to experience significant emotional engagement with museum resources. The smart model also gave BPS users a level of control over the AD which gave them a greater sense of empowerment and independence, which is particularly important for BPS visitors with varying sight conditions. We conclude that our SJSM approach has considerable potential as an approach to universal access, and to increase emotional engagement with museum collections. We also propose several developments which could further extend the approach and its implementation.

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

scholarly journals Use of Quantitative Molecular Diagnostic Assays to Investigate Fusarium Dry Rot in Potato Stocks and Soil

2005 ◽  
Vol 95 (12) ◽  
pp. 1462-1471 ◽  
Author(s):  
D. W. Cullen ◽  
I. K. Toth ◽  
Y. Pitkin ◽  
N. Boonham ◽  
K. Walsh ◽  
...  
Keyword(s):  
Control Strategies ◽  
Disease Incidence ◽  
Harvest Date ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Fusarium Spp ◽  
Dry Rot ◽  
Diagnostic Assays ◽  
Final Harvest

Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 104, 105, and 106 conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 104 conidia/ml for F. sulphureum and 105 conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.

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