A novel molecular diagnostic method for the gut content analysis of Philaenus DNA

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Isabel Rodrigues ◽  
Vítor Ramos ◽  
Jacinto Benhadi-Marín ◽  
Aránzazu Moreno ◽  
Alberto Fereres ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Control Strategies ◽  
Control Measure ◽  
Coi Gene ◽  
Molecular Diagnostic ◽  
Generalist Predators ◽  
Specificity And Sensitivity ◽  
Vector Borne ◽  
Polymerase Chain ◽  
Primer Sets

AbstractPhilaenus spumarius is a vector of Xylella fastidiosa, one of the most dangerous plants pathogenic bacteria worldwide. There is currently no control measure against this pathogen. Thus, the development of vector control strategies, like generalist predators, such as spiders, could be essential to limit the spread of this vector-borne pathogen. In this study, a polymerase chain reaction (PCR)-based approach was developed to principally detect DNA of P. spumarius in the spider’s gut. Accordingly, 20 primer pairs, targeting the mitochondrial cytochrome oxidase I (COI) and cytochrome b (cytB) genes, were tested for specificity, sensitivity, and efficiency in detecting P. spumarius DNA. Overall, two primer sets, targeting COI gene (COI_Ph71F/COI_Ph941R) and the cytB gene (cytB_Ph85F/cytB_Ph635R), showed the highest specificity and sensitivity, being able to amplify 870 pb and 550 bp fragments, respectively, with P. spumarius DNA concentrations 100-fold lower than that of the DNA of non-target species. Among these two primer sets, the cytB_Ph85F/cytB_Ph635R was able to detect P. spumarius in the spider Xysticus acerbus, reaching 50% detection success 82 h after feeding. The feasibility of this primer set to detect predation of P. spumarius by spiders was confirmed in the field, where 20% of the collected spiders presented positive amplifications.

scholarly journals Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

2000 ◽  
Vol 90 (10) ◽  
pp. 1112-1119 ◽  
Author(s):  
Howard S. Judelson ◽  
Paul W. Tooley
Keyword(s):  
Polymerase Chain Reaction ◽  
Phytophthora Infestans ◽  
Positive Control ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Diagnostic Assays ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Diagnostic Applications

Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.

scholarly journals Fast and Accurate Large-Scale Detection of β-Lactamase Genes Conferring Antibiotic Resistance

2015 ◽  
Vol 59 (10) ◽  
pp. 5967-5975 ◽  
Author(s):  
Jae Jin Lee ◽  
Jung Hun Lee ◽  
Dae Beom Kwon ◽  
Jeong Ho Jeon ◽  
Kwang Seung Park ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
Large Scale ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Clinical Isolates ◽  
Molecular Diagnostic ◽  
Resistant Bacteria ◽  
Specificity And Sensitivity ◽  
Almost All

ABSTRACTFast detection of β-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limitedblagene types, these methods have significant limitations, such as their failure to detect almost all clinically availableblagenes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scalebladetection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of thelarge-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, thelarge-scaleblaFinder detected almost all clinically availableblagenes. Notably, thelarge-scaleblaFinder detected 24 additional unreportedblagenes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types ofblagenes can miss unexpectedblagenes existing in pathogenic bacteria, and our method has the ability to detect almost allblagenes existing in a clinical isolate. The ability oflarge-scaleblaFinder to detectblagenes on a large scale enables prompt application to the detection of almost allblagenes present in bacterial pathogens. The widespread use of thelarge-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination ofblagenes and minimizing the spread of resistant bacteria.

scholarly journals ISOLASI NON-DESTRUKTIF DAN DESTRUKTIF GEN COI PADA SERANGGA JENIS COLEOPTERA PEMBAWA PATOGEN CERATOCYSTIS

2021 ◽  
Vol 15 (1) ◽  
pp. 1-11
Author(s):  
Anindya Safita Ningtias ◽  
◽  
Istiana Prihatini ◽  
Maryatul Qiptiyah
Keyword(s):  
Species Identification ◽  
Pathogenic Fungi ◽  
Coi Gene ◽  
Chain Reaction ◽  
Crucial Step ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Non Destructive ◽  
Destructive Methods ◽  
Molecular Characters

Identification of insect species using molecular approach is one of the first steps in managing Ceratocystis. Proper species identification of the vectorsof Ceratocystiswill provide an effective way in limiting the distribution of this pathogenic fungi. The isolation of insect DNA is a crucial step in species identification using molecular characters. The aim of this research was to obtain the most effective method for isolating the COI gene and to confirm the insect DNA using amplification of the DNA through polymerase chain reaction (PCR). Twelve unidentified Coleoptera specimen collected from Acacia spp. plantation in Pelalawan Riau were randomly selected for this study. The insect DNA were isolated using the CTAB buffer with four different pre-incubation treatments, involved a non destructive method, i.e. soaking (R), destructive methods, i.e. crushing (H), soaking-crushing (RH) and freezing-crushing (FH). Two sets of primer, LCO1490/HCO2196 and LCO1490/HCO2198 were used to amplify the DNA of COI gene. The results shows, the COI gene was isolated from all pre-incubation treatments, except in the non-destructive treatment. The isolated DNA of COI gene was successfully amplified using both primer sets used in this study

Improving the quality of bacteriological studies for infections caused by carbapenem resistant Klebsiella pneumoniae using molecular diagnostic methods

2020 ◽  
pp. 6-17
Author(s):  
A. Chizhikova ◽  
E. Lisitsyna
Keyword(s):  
Klebsiella Pneumoniae ◽  
Pathogenic Bacteria ◽  
Test System ◽  
Ease Of Use ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Fast Analysis ◽  
Carbapenem Resistant ◽  
Specificity And Sensitivity

The resistance of gram-negative pathogenic bacteria to antibiotics of the β-lactam group — carbapenems — is associated with the ability of bacteria to produce carbapenemases. The relevant task is to determine carbapenem-resistant microorganisms to control the spread of resistant strains and conduct effective pharmacotherapy. The results of the development of molecular diagnostic methods, including use of polymerase chain reaction (PCR), for the detection of carbapenem resistant Klebsiella pneumoniae bacteria are presented. A multiplex PCR test system was developed for detecting carbapenem-resistant K. pneumoniae producing KPC and OXA 48-like carbapenemases. The test system is characterized by high specificity and sensitivity, ease of use, fast analysis time (up to 3 hours). Its introduction into clinical and diagnostic practice is promising in terms of improving the quality of bacteriological studies.

scholarly journals Using a Genome-Based PCR Primer Prediction Pipeline to Develop Molecular Diagnostics for the Turfgrass Pathogen Acidovorax avenae

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2224-2232 ◽  
Author(s):  
Paul R. Giordano ◽  
Jie Wang ◽  
Joseph M. Vargas ◽  
Janette Jacobs ◽  
Martin I. Chilvers ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Creeping Bentgrass ◽  
Pcr Assay ◽  
Putting Greens ◽  
Specificity And Sensitivity ◽  
A Genome ◽  
Polymerase Chain ◽  
Primer Sets

Acidovorax avenae is the causal agent of bacterial etiolation and decline (BED) of creeping bentgrass, a poorly understood and often misdiagnosed disease that can result in considerable aesthetic and functional damage to golf course putting greens. Current diagnostics of BED are based on laborious culture-based methods. In this work, we employed a novel alignment-free primer prediction pipeline to design diagnostic primers for turfgrass-pathogenic A. avenae using 15 draft genomes of closely related target and nontarget Acidovorax spp. as input. Twenty candidate primer sets specific to turfgrass-pathogenic A. avenae were designed. The specificity and sensitivity of these primer sets were validated via a traditional polymerase chain reaction (PCR) and a real-time PCR assay. Primer sets 0017 and 0019 coupled with an internal oligo probe showed optimal sensitivity and specificity when evaluated with the target pathogen, closely related bacterial species, and microorganisms that inhabit the same host and soil environment. Finally, the accuracy of the newly developed real-time PCR assay was evaluated to detect BED pathogens from BED-symptomatic and asymptomatic turfgrass samples. The diagnostic results produced by the real-time PCR assay were consistent with results of a cultural-based method. This assay will allow quicker and more effective detection of the BED pathogen, thus potentially reducing misdiagnoses and unnecessary usage of fungicides.

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 Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Elton J. R. Vasconcelos ◽  
Chayan Roy ◽  
Joseph A. Geiger ◽  
Kristina M. Oney ◽  
Melody Koo ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Complete Analysis ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Spotted Fever Group ◽  
Next Generation ◽  
Vector Borne ◽  
16 S Rrna ◽  
Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

scholarly journals Wolbachia: endosymbiont of onchocercid nematodes and their vectors

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Ranju Ravindran Santhakumari Manoj ◽  
Maria Stefania Latrofa ◽  
Sara Epis ◽  
Domenico Otranto
Keyword(s):  
Control Strategies ◽  
Molecular Data ◽  
Scientific Data ◽  
Growth And Survival ◽  
Vector Borne Diseases ◽  
Wide Range ◽  
Veterinary Importance ◽  
Wolbachia Endosymbiont ◽  
Vector Borne ◽  
And Control

Abstract Background Wolbachia is an obligate intracellular maternally transmitted, gram-negative bacterium which forms a spectrum of endosymbiotic relationships from parasitism to obligatory mutualism in a wide range of arthropods and onchocercid nematodes, respectively. In arthropods Wolbachia produces reproductive manipulations such as male killing, feminization, parthenogenesis and cytoplasmic incompatibility for its propagation and provides an additional fitness benefit for the host to protect against pathogens, whilst in onchocercid nematodes, apart from the mutual metabolic dependence, this bacterium is involved in moulting, embryogenesis, growth and survival of the host. Methods This review details the molecular data of Wolbachia and its effect on host biology, immunity, ecology and evolution, reproduction, endosymbiont-based treatment and control strategies exploited for filariasis. Relevant peer-reviewed scientic papers available in various authenticated scientific data bases were considered while writing the review. Conclusions The information presented provides an overview on Wolbachia biology and its use in the control and/or treatment of vectors, onchocercid nematodes and viral diseases of medical and veterinary importance. This offers the development of new approaches for the control of a variety of vector-borne diseases. Graphic Abstract

scholarly journals High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 293
Author(s):  
Idalécia Cossa-Moiane ◽  
Hermínio Cossa ◽  
Adilson Fernando Loforte Bauhofer ◽  
Jorfélia Chilaúle ◽  
Esperança Lourenço Guimarães ◽  
...  
Keyword(s):  
Public Hospitals ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
P Value ◽  
Cryptosporidium Hominis ◽  
Cryptosporidium Spp ◽  
Pcr Rflp ◽  
Maputo City ◽  
Stool Samples ◽  
Polymerase Chain

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

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