scholarly journals Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

Forests ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 479
Author(s):  
Chiara Aglietti ◽  
Colton D. Meinecke ◽  
Luisa Ghelardini ◽  
Irene Barnes ◽  
Ariska van der Nest ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Morphological Characteristics ◽  
Lamp Assay ◽  
High Specificity ◽  
Portable Devices ◽  
Forest Protection ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Lecanosticta Acicola ◽  
Dothistroma Pini

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.

scholarly journals Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP)

2021 ◽  
Vol 18 (9) ◽  
pp. 4401
Author(s):  
Yufei Chen ◽  
Hao Li ◽  
Liu Yang ◽  
Lei Wang ◽  
Ruyi Sun ◽  
...  
Keyword(s):  
Rapid Detection ◽  
High Throughput Screening ◽  
Clostridium Botulinum ◽  
Lamp Assay ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Target Dna

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

scholarly journals Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP)

2021 ◽  
Author(s):  
Johannes Köck ◽  
Christoph Gottschalk ◽  
Sebastian Ulrich ◽  
Karin Schwaiger ◽  
Manfred Gareis ◽  
...  
Keyword(s):  
Lamp Assay ◽  
High Specificity ◽  
Neutral Red ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Visual Signal ◽  
Stachybotrys Chartarum ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Macrocyclic Trichothecene

AbstractCytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay’s detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

scholarly journals Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

Forests ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 362
Author(s):  
Colin L. Myrholm ◽  
Bradley D. Tomm ◽  
Renate Heinzelmann ◽  
Nicolas Feau ◽  
Richard C. Hamelin ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Pine Needle ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Lecanosticta Acicola ◽  
Dothistroma Pini ◽  
Biology Laboratory ◽  
Dothistroma Septosporum ◽  
Selection Of

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

scholarly journals Development of a triplex real-time PCR for simultaneous detection of allergenic ingredients in processed food

2018 ◽  
Vol 36 (No. 1) ◽  
pp. 22-27 ◽  
Author(s):  
Wenju Zhang ◽  
Yulei Zhao ◽  
Qingjin Xu ◽  
Qin Chen
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Processed Food ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Primer Sets ◽  
Specific Pcr Primer ◽  
Suitable System

SYBR Green real-time or quantitative PCR (Q-PCR) is a suitable system in which to establish a multiplex method to detect allergenic ingredients in food. In this study, a triplex Q-PCR method was developed to detect trace amounts of peanut, soybean and sesame in processed food. Specific PCR primer sets were designed and the concentration of the primers used in the triplex PCR was optimised. The triplex method showed high specificity and sensitivity which were similar to those of the simplex method, and it was applied for the detection of allergenic ingredients in commercially available processed food. The results demonstrate that the developed triplex Q-PCR is a quick, reliable and efficient method for the detection of allergenic ingredients in processed food.

scholarly journals A LAMP at the end of the tunnel: a rapid, field deployable assay for the kauri dieback pathogen,Phytophthora agathidicida

2019 ◽  
Author(s):  
Richard C. Winkworth ◽  
Briana C.W. Nelson ◽  
Stanley E. Bellgard ◽  
Chantal M. Probst ◽  
Patricia A. McLenachan ◽  
...  
Keyword(s):  
Hybrid Approach ◽  
Lamp Assay ◽  
High Specificity ◽  
Cost Effective ◽  
Direct Evaluation ◽  
Term Survival ◽  
Apocytochrome B ◽  
Specificity And Sensitivity ◽  
Laboratory Facility ◽  
Assay Performance

AbstractThe collar rot causing oomycete,Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14-20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection ofP. agathidicidathat targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of otherPhytophthoraisolates and detected as little as 1 fg of totalP. agathidicidaDNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended bioassay and LAMP testing of DNA extracted from baits. In these comparisons,P. agathidicidawas detected more frequently using the LAMP assay. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions aboutP. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid assay also has the potential to empower local communities. These communities could use this diagnostic tool to evaluate the pathogen status of local kauri stands, providing information around which to base their management and allowing informed engagement with wider initiatives.

scholarly journals An Editing-Site-Specific PCR Method for Detection and Quantification of CAO1-Edited Rice

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1209
Author(s):  
Hongwen Zhang ◽  
Jun Li ◽  
Shengbo Zhao ◽  
Xiaohong Yan ◽  
Nengwu Si ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
High Specificity ◽  
Editing Site ◽  
Locked Nucleic Acid ◽  
Molecular Characteristics ◽  
Site Specific ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Detection And Quantification

Genome-edited plants created by genome editing technology have been approved for commercialization. Due to molecular characteristics that differ from classic genetically modified organisms (GMOs), establishing regulation-compliant analytical methods for identification and quantification of genome-edited plants has always been regarded as a challenging task. An editing-site-specific PCR method was developed based on the unique edited sequence in CAO1-edited rice plants. Test results of seven primer/probe sets indicated that this method can identify specific CAO1-edited rice from other CAO1-edited rice and wild types of rice with high specificity and sensitivity. The use of LNA (locked nucleic acid) in a probe can efficiently increase the specificity of the editing-site-specific PCR method at increased annealing temperature which can eliminate non-specific amplification of the non-target. The genome-edited ingredient content in blinded samples at the level of 0.1% to 5.0% was accurately quantified by this method on the ddPCR platform with RSD of <15% and bias in the range of ±17%, meeting the performance requirements for GMO detection method. The developed editing-site-specific PCR method presents a promising detection and quantification technique for genome-edited plants with known edited sequence.

Methods for the Quantification of Salivary Cortisol and of a-amylase in Biosensors and Portable Devices

2018 ◽  
Vol 68 (12) ◽  
pp. 2857-2859
Author(s):  
Cristina Mihaela Ghiciuc ◽  
Andreea Silvana Szalontay ◽  
Luminita Radulescu ◽  
Sebastian Cozma ◽  
Catalina Elena Lupusoru ◽  
...  
Keyword(s):  
Real Time ◽  
Medical Practice ◽  
Salivary Cortisol ◽  
Clinical Studies ◽  
Large Scale ◽  
Psychological Research ◽  
Portable Devices ◽  
Salivary Amylase ◽  
Specificity And Sensitivity

There is an increasing interest in the analysis of salivary biomarkers for medical practice. The objective of this article was to identify the specificity and sensitivity of quantification methods used in biosensors or portable devices for the determination of salivary cortisol and salivary a-amylase. There are no biosensors and portable devices for salivary amylase and cortisol that are used on a large scale in clinical studies. These devices would be useful in assessing more real-time psychological research in the future.

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

scholarly journals Identification of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 202
Author(s):  
Jianghua Chen ◽  
Zihang Zhu ◽  
Yanping Fu ◽  
Jiasen Cheng ◽  
Jiatao Xie ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Citrus Fruit ◽  
Economic Loss ◽  
Fruit Rot ◽  
Post Inoculation ◽  
Citrus Reticulata ◽  
Postharvest Diseases ◽  
Citrus Reticulata Blanco

Considering the huge economic loss caused by postharvest diseases, the identification and prevention of citrus postharvest diseases is vital to the citrus industry. In 2018, 16 decayed citrus fruit from four citrus varieties—Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)—showing soft rot and sogginess on their surfaces and covered with white mycelia were collected from storage rooms in seven provinces. The pathogens were isolated and the pathogenicity of the isolates was tested. The fungal strains were identified as Lasiodiplodia pseudotheobromae based on their morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin (TUB) gene sequences. The strains could infect wounded citrus fruit and cause decay within two days post inoculation, but could not infect unwounded fruit. To our knowledge, this is the first report of citrus fruit decay caused by L. pseudotheobromae in China.

scholarly journals SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikail Dogan ◽  
Lina Kozhaya ◽  
Lindsey Placek ◽  
Courtney Gunter ◽  
Mesut Yigit ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
High Specificity ◽  
Spike Protein ◽  
Humoral Immune ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Specific Igg ◽  
Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Sign in / Sign up

Export Citation Format

Share Document