scholarly journals A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Arati Agarwal ◽  
J. Paul Cunningham ◽  
Isabel Valenzuela ◽  
Mark J. Blacket
Keyword(s):  
Insect Pest ◽  
Extraction Procedure ◽  
Dna Barcode ◽  
Lamp Assay ◽  
Cost Effective ◽  
Daktulosphaira Vitifoliae ◽  
Synthetic Dna ◽  
False Positive Identification ◽  
Containment Measures ◽  
Molecular Identification Methods

AbstractGrape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alisa Alekseenko ◽  
Donal Barrett ◽  
Yerma Pareja-Sanchez ◽  
Rebecca J. Howard ◽  
Emilia Strandback ◽  
...  
Keyword(s):  
Large Scale ◽  
Direct Detection ◽  
Dna Polymerases ◽  
Scale Up ◽  
Lamp Assay ◽  
Cost Effective ◽  
Reaction Conditions ◽  
Clinical Patient ◽  
Reverse Transcriptases ◽  
Large Scale Testing

AbstractRT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Cucumber vs Ants: a Case Against the Myth of the Uses of Plant Extracts in Insect Pest Management

Sociobiology ◽  
2021 ◽  
Vol 68 (2) ◽  
pp. 5813
Author(s):  
Matan Shelomi ◽  
Bo-Jun Qiu ◽  
Lin-Ting Huang
Keyword(s):  
Plant Extracts ◽  
Scientific Evidence ◽  
Insect Pest ◽  
Management Strategies ◽  
Cost Effective ◽  
Detectable Effect ◽  
Realistic Situation ◽  
Potential Impact ◽  
High Concentrations ◽  
Ant Repellent

An accumulation of questionable scientific reports on the use of natural plant extracts to control household pest insects, using biologically irrelevant experimental designs and extremely high concentrations, has resulted in a publication bias: “promising” studies claiming readily available plants can repel various insects, including social insects, despite no usable data to judge cost-effectiveness or sustainability in a realistic situation. The Internet provides a further torrent of untested claims, generating a background noise of misinformation. An example is the belief that cucumbers are “natural” ant repellent, widely reported in such informal literature, despite no direct evidence for or against this claim. We tested this popular assertion using peel extracts of cucumber and the related bitter melon as olfactory and gustatory repellents against ants. Extracts of both fruit peels in water, methanol, or hexane were statistically significant but effectively weak gustatory repellents. Aqueous cucumber peel extract has a significant but mild olfactory repellent effect: about half of the ants were repelled relative to none in a control. While the myth may have a grain of truth to it, as cucumber does have a mild but detectable effect on ants in an artificial setup, its potential impact on keeping ants out of a treated perimeter would be extremely short-lived and not cost-effective. Superior ant management strategies are currently available. The promotion of “natural” products must be rooted in scientific evidence of a successful and cost-effective implementation prospect.

scholarly journals Indigenous Technical Knowledge in Plant Disease Management

2021 ◽  
pp. 377-380
Author(s):  
Hari Prasanna Sahu ◽  
Rakesh Roshan Satapathy
Keyword(s):  
Disease Management ◽  
Plant Disease ◽  
Insect Pest ◽  
Cost Effective ◽  
Technical Knowledge ◽  
Harvest Management ◽  
Soil Fertility Management ◽  
Sustainable Environment ◽  
Indigenous Technical Knowledge ◽  
Plant Disease Management

The origins of indigenous technical/traditional knowledge are local, rural &community. It's utilised in forecasting of weather for better seed germination, soil, water, and soil fertility management, disease and insect pest control of plants & animals, and post-harvest management, among other things. India has a variety of indigenous agricultural practises which are still popular in organic agriculture in India's many states and are sustainable, environment friendly, profitable, and cost-effective. This review paper contains an overview of Indigenous Technical Knowledge in Plant Disease Management to help researchers in the future.

scholarly journals Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrew Currin ◽  
Neil Swainston ◽  
Mark S Dunstan ◽  
Adrian J Jervis ◽  
Paul Mulherin ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequencing ◽  
Cost Effective ◽  
Polymorphism Analysis ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Design Build ◽  
Hardware Costs

Abstract Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

scholarly journals Determination of Cr (VI) Content in Water-Soluble Dyes

2020 ◽  
Vol 59 (1) ◽  
pp. 88-94
Author(s):  
M P Sathianarayanan ◽  
Rina Nayak ◽  
Yogesh Hande
Keyword(s):  
Spectroscopic Analysis ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Load ◽  
Water Soluble ◽  
Recovery Study ◽  
Good Repeatability ◽  
Repeatability And Reproducibility ◽  
Standard Solution

Abstract Hexavalent chromium detection in the presence of high load of colorants is a challenge, and it is an important area of study. Colorants are a class of interfering substance in many spectroscopic analysis and chromatographic separation and detection. In this study, a method has been developed to separate out Cr (VI) and water-soluble dyes by using activated charcoal as an absorption medium. The extraction procedure was optimized with Cr (VI) standard solution for quantification. The efficacy of the extraction procedure for the removal of water-soluble dyes and detection of Cr (VI) was checked with a spike recovery study. Based on the spike recovery study, the method has been validated as per the international validation protocol. The method is simple, cost effective and has a detection limit down up to 3.0 mg/kg. The recovery rate of Cr (VI) in water-soluble dyes like reactive yellow HE 6G, reactive red 218, turquoise blue HGN, reactive navy blue RX and reactive black 5A was found to be more than 90% with a good repeatability and reproducibility.

scholarly journals Loop-mediated isothermal amplification (LAMP) : A rapid molecular diagnosis technique for detection of human pathogens

2017 ◽  
Vol 07 (03) ◽  
pp. 042-048
Author(s):  
Gunimala Chakraborty ◽  
Indrani Karunasagar ◽  
Anirban Chakraborty
Keyword(s):  
Treatment Strategies ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Current Status ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Pathogens ◽  
Quality Healthcare ◽  
Loop Mediated Isothermal Amplification

AbstractDelivery of quality healthcare in case of an infectious disease depends on how efficiently and how quickly the responsible pathogens are detected from the samples. Molecular methods can detect the presence of pathogens in a rapid and sensitive manner. Over the years, a number of such assays have been developed. However, these methods, although highly reliable and efficient, require use of expensive equipment, reagents, and trained personnel. Therefore, development of molecular assays that are simple, rapid, cost-effective, yet sensitive, is highly warranted to ensure efficient management or treatment strategies. Loop-mediated isothermal amplification (LAMP), a technique invented in the year 2000, is a novel method that amplifies DNA at isothermal conditions. Since its invention, this technique has been one of the most extensively used molecular diagnostic tools in the field of diagnostics offering rapid, accurate and cost-effective diagnosis of infectious diseases. Using the LAMP principle, many commercial kits have been developed in the last decade for a variety of human pathogens including bacteria, viruses and parasites. Currently LAMP assay is being considered as an effective diagnostic tool for use in developing countries because of its simple working protocol, allowing even an onsite application. The focus of this review is to describe the salient features of this technique the current status of development of LAMP assays with an emphasis on the pathogens of clinical significance.

scholarly journals Artificial Intelligence-Assisted Loop Mediated Isothermal Amplification (AI-LAMP) for Rapid Detection of SARS-CoV-2

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 972 ◽  
Author(s):  
Mohammed A. Rohaim ◽  
Emily Clayton ◽  
Irem Sahin ◽  
Julianne Vilela ◽  
Manar E. Khalifa ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
De Novo ◽  
Tracking System ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Analytical Sensitivity ◽  
Diagnostic Device ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.

scholarly journals Using a Loop-Mediated Isothermal Amplification (LAMP) Assay and Direct DNA Extraction Method from Wood for Rapid Detection of Verticillium dahliae in Olive Trees

2017 ◽  
Vol 14 (2) ◽  
pp. 727-734
Author(s):  
Saba Aslani ◽  
Ghasemali Garoosi ◽  
Hossein Jafary
Keyword(s):  
Dna Extraction ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Lamp Assay ◽  
Cost Effective ◽  
Pathogenic Fungi ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Minimum Requirements ◽  
Olive Trees

ABSTRACT: Verticillium wilt, which is caused by the fungus Verticillium dahliae, is one of the most important olive diseases worldwide. There are many ways to extract DNA from plant pathogenic fungi and from plant tissues for molecular-based diagnostic assays. LAMP is a new and sensitive molecular-based technique used for detection of plant pathogenic agents with minimum requirements needed. In this study, we tried to achieve a simple, cost effective and efficient method of DNA extraction from both Verticillium dahliae fungus and from infected wood samples in order to run a loop-mediated isothermal amplification (LAMP) assay. Efficiency of three DNA isolation methods from both mycelia and infected wood samples was evaluated. For this purpose, wood samples from infected olive trees were collected from Tarom region in Zanjan province and the samples were cultured on the media. The fungus was isolated and identified as V. dahliae based on morphological features. Then the genomic DNA was extracted using traditional CTAB method, fast NaOH method and direct isolation method from infected wood samples. After assessment of the quality and the quantity of the extracted DNA samples, a LAMP assay was ran using specific primer pairs and the DNA templates extracted using three different methods. In spite of the significant differences in the quantity of DNA samples, LAMP assay could successfully detect the fungus in all samples. The improved direct isolation of the DNA of V. dahlia from infected wood, followed by a LAMP assay could considerably shortened the detection process of the fungus and hence is a suitable method for screening of olive trees and saplings against Verticillium wilt disease.

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