Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay

Citrus is the most extensively produced fruit tree crop in the world and is grown in over 130 countries. Fungal diseases in citrus can cause significant losses in yield and quality. An accurate diagnosis is critical for determining the best management practices and preventing future losses. In this study, a Recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas) system was established with the integration of a lateral flow assay (LFA) readout system for diagnosis of citrus scab. This detection can be completed within 1 h, is highly sensitive and prevents cross-reactions with other common fungal citrus diseases. Furthermore, the detection system is compatible with crude DNA extracted from infected plant tissue. This RPA-CRISPR/Cas12a-LFA system provides a sensitive, rapid, and cost-effective method with promising and significant practical value for point-of-care diagnosis of citrus scab. To our knowledge, this is the first report to establish an RPA- and CRISPR-based method with LFA for fungal diseases in plants.

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A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

Journal of Clinical and Medical Research ◽

10.37191/mapsci-2582-4333-2(3)-032 ◽

2020 ◽

Author(s):

Carla Eiras

Keyword(s):

Interleukin 6 ◽

Limit Of Detection ◽

Inflammatory Diseases ◽

Point Of Care ◽

Colorimetric Detection ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Aggregation Assay ◽

Before And After ◽

Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM).The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

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A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

Journal of Clinical and Medical Research ◽

10.37191/mapsci-2582-4333-2(2)-032 ◽

2020 ◽

Author(s):

Carla Eiras

Keyword(s):

Interleukin 6 ◽

Limit Of Detection ◽

Inflammatory Diseases ◽

Point Of Care ◽

Colorimetric Detection ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Aggregation Assay ◽

Before And After ◽

Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated at a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM). The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

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Are Semi-Quantitative Clinical Cultures Inadequate? Comparison to Quantitative Analysis of 1053 Bacterial Isolates from 350 Wounds

Diagnostics ◽

10.3390/diagnostics11071239 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1239

Author(s):

Thomas E. Serena ◽

Philip G. Bowler ◽

Gregory S. Schultz ◽

Anna D’souza ◽

Monique Y. Rennie

Keyword(s):

Quantitative Analysis ◽

Best Management Practices ◽

Management Practices ◽

Chronic Wounds ◽

Point Of Care ◽

Bacterial Load ◽

Quantitative Culture ◽

Simple Method ◽

Point Of Care Technology ◽

Care Technology

Early awareness and management of bacterial burden and biofilm is essential to wound healing. Semi-quantitative analysis of swab or biopsy samples is a relatively simple method for measuring wound microbial load. The accuracy of semi-quantitative culture analysis was compared to ‘gold standard’ quantitative culture analysis using 428 tissue biopsies from 350 chronic wounds. Semi-quantitative results, obtained by serial dilution of biopsy homogenates streaked onto culture plates divided into 4 quadrants representing occasional, light, moderate, and heavy growth, were compared to total bacterial load quantified as colony-forming units per gram (CFU/g). Light growth, typically considered an insignificant finding, averaged a clinically significant 2.5 × 105 CFU/g (SE = 6.3 × 104 CFU/g). Occasional growth (range: 102–106 CFU/g) and light growth (103–107 CFU/g) corresponded to quantitative values that spanned a 5-log range; moderate and heavy growth corresponded to a range of 4-log and 6-log, respectively, with a high degree of overlap in range of CFU/g per category. Since tissue biopsy and quantitative culture cannot be widely practiced and semi-quantitative analysis is unreliable, other clinically relevant approaches are required to determine wound bioburden and guide best management practices. Fluorescence imaging is a point-of-care technology that offers great potential in this field.

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Lateral Flow Assay Based on Paper-Hydrogel Hybrid Material for Sensitive Point-of-Care Detection of Dengue Virus

Advanced Healthcare Materials ◽

10.1002/adhm.201600920 ◽

2016 ◽

Vol 6 (1) ◽

pp. 1600920 ◽

Cited By ~ 34

Author(s):

Jane Ru Choi ◽

Kar Wey Yong ◽

Ruihua Tang ◽

Yan Gong ◽

Ting Wen ◽

...

Keyword(s):

Dengue Virus ◽

Hybrid Material ◽

Point Of Care ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Point Of Care Detection

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Lateral flow assays for the detection of African swine fever virus antigen are not fit for field diagnosis of wild boar carcasses

10.22541/au.161994432.24828107/v1 ◽

2021 ◽

Author(s):

Paul Deutschmann ◽

Jutta Pikalo ◽

Martin Beer ◽

Sandra Blome

Keyword(s):

Wild Boar ◽

Point Of Care ◽

African Swine Fever ◽

Virus Antigen ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Field Conditions ◽

Real Field ◽

Data Set ◽

Field Samples

African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, “point-of-care” (POC) tests have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. Along these lines, the INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) promises virus antigen detection under field conditions within minutes. In the present study, we evaluated the performance of the assay with selected high-quality reference blood samples, and also with real field samples from wild boar carcasses in different stages of decay from the ongoing ASF outbreak in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct carcass testing without any modification resulted in a drastically reduced sensitivity of only 12.5%. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of carcass samples. A diagnostic specificity of 100% was observed. However, most of the German ASF cases in wild boar would have been missed using the lateral flow assay (LFA) alone. Therefore, the antigen-specific LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen.

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A Self-Contained Chemiluminescent Lateral Flow Assay for Point-of-Care Testing

Analytical Chemistry ◽

10.1021/acs.analchem.8b01543 ◽

2018 ◽

Vol 90 (15) ◽

pp. 9132-9137 ◽

Cited By ~ 27

Author(s):

Jinqi Deng ◽

Mingzhu Yang ◽

Jing Wu ◽

Wei Zhang ◽

Xingyu Jiang

Keyword(s):

Point Of Care ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Point Of Care Testing

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A first of its kind quantitative functional C1-esterase inhibitor lateral flow assay for hereditary angioedema point-of-care diagnostic testing

International Immunopharmacology ◽

10.1016/j.intimp.2020.106526 ◽

2020 ◽

Vol 83 ◽

pp. 106526

Author(s):

Priya S. Chockalingam ◽

Zhiwei Zhou ◽

Brijesh K. Garg ◽

Hans Boehringer ◽

Roy Chung ◽

...

Keyword(s):

Hereditary Angioedema ◽

Point Of Care ◽

Diagnostic Testing ◽

Lateral Flow ◽

Lateral Flow Assay ◽

C1 Esterase Inhibitor ◽

Esterase Inhibitor

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Multi-Objective Optimization for Selecting and Siting the Cost-Effective BMPs by Coupling Revised GWLF Model and NSGAII Algorithm

Water ◽

10.3390/w12010235 ◽

2020 ◽

Vol 12 (1) ◽

pp. 235 ◽

Cited By ~ 1

Author(s):

Zuoda Qi ◽

Gelin Kang ◽

Xiaojin Wu ◽

Yuting Sun ◽

Yuqiu Wang

Keyword(s):

Best Management Practices ◽

Management Practices ◽

Cost Effective ◽

Control Measures ◽

Multi Objective Optimization ◽

Watershed Model ◽

Filter Strips ◽

Multi Objective ◽

Dissolved Nitrogen ◽

The Cost

Best management practices (BMPs) are an effective way to control water pollution. However, identification of the optimal distribution and cost-effect of BMPs provides a great challenge for watershed policy makers. In this paper, a semi-distributed, low-data, and robust watershed model, the Revised Generalized Watershed Loading Function (RGWLF), is improved by adding the pollutant attenuation process in the river channel and a bank filter strips reduction function. Three types of pollution control measures—point source wastewater treatment, bank filter strips, and converting farmland to forest—are considered, and the cost of each measure is determined. Furthermore, the RGWLF watershed model is coupled with a widely recognized multi-objective optimization algorithm, the non-dominated sorting genetic algorithm II (NSGAII), the combination of which is applied in the Luanhe watershed to search for spatial BMPs for dissolved nitrogen (DisN). Fifty scenarios were finally selected from numerous possibilities and the results indicate that, at a minimum cost of 9.09 × 107 yuan, the DisN load is 3.1 × 107 kg and, at a maximum cost of 1.77 × 108 yuan, the total dissolved nitrogen load is 1.31 × 107 kg; with the no-measures scenario, the DisN load is 4.05 × 107 kg. This BMP optimization model system could assist decision-makers in determining a scientifically comprehensive plan to realize cost-effective goals for the watershed.

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