scholarly journals Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

2019 ◽  
Author(s):  
Umer Chaudhry ◽  
Qasim Ali ◽  
Imran Rashid ◽  
Muhammad Zubair Shabbir ◽  
Muhammad Abbas ◽  
...  
Keyword(s):  
Species Composition ◽  
Species Interactions ◽  
Control Strategies ◽  
Detection Threshold ◽  
Amplicon Sequencing ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Parasitic Infections ◽  
Sequencing Platform ◽  
Field Samples

AbstractPiroplasmosis is caused by tick-borne haemoprotozoa of the generaTheileriaandBabesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities.TheileriaandBabesiavary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a “haemoprotobiome”. First, four phenotypically-verified species ofTheileriaandBabesiawere used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The “haemoprotobiome” concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

scholarly journals Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257971
Author(s):  
David A. Wilkinson ◽  
Matthew Edwards ◽  
Jackie Benschop ◽  
Shahista Nisa
Keyword(s):  
New Zealand ◽  
Diagnostic Techniques ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pathogenic Leptospira ◽  
Molecular Barcoding ◽  
Sequencing Platform ◽  
Oxford Nanopore ◽  
Culture Independent ◽  
Variant Identification

Leptospirosis is a zoonotic disease of global importance. The breadth of Leptospira diversity associated with both human and animal disease poses major logistical challenges to the use of classical diagnostic techniques, and increasingly molecular diagnostic tools are used for their detection. In New Zealand, this has resulted in an increase in positive cases reported nationally that have not been attributed to the infecting serovar or genomospecies. In this study, we used data from all pathogenic Leptospira genomes to identify a partial region of the glmU gene as a suitable locus for the discrimination of the infecting species and serovars of New Zealand-endemic Leptospira. This method can be used in culture and culture-independent scenarios making it flexible for diagnostics in humans, animals, and environmental samples. We explored the use of this locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION. Sequences obtained by this method allowed specific identification of Leptospira species in mixed and enriched environmental cultures, however read error inherent in the MinION sequencing system reduced the accuracy of strain/variant identification. Using this approach to characterise Leptospira in enriched environmental cultures, we detected the likely presence of Leptospira genomospecies that have not been reported in New Zealand to date. This included a strain of L. borgpetersenii that has recently been identified in dairy cattle and sequences similar to those of L. mayottensis. L. tipperaryensis, L. dzianensis and L. alstonii.

scholarly journals A diagnostic multiplex PCR scheme for identification of plant-associated bacteria of the genusPantoea

2018 ◽  
Author(s):  
Kossi Kini ◽  
Raoul Agnimonhan ◽  
Rachelle Dossa ◽  
Drissa Silué ◽  
Ralf Koebnik
Keyword(s):  
Multiplex Pcr ◽  
Plant Protection ◽  
Detection Threshold ◽  
Leaf Blight ◽  
Crop Plants ◽  
Plant Diseases ◽  
Bacterial Leaf Blight ◽  
Epidemiological Surveillance ◽  
Molecular Diagnostic ◽  
Diagnostic Tools

AbstractBackgroundThe genusPantoeaforms a complex of more than 25 species, among which several cause diseases of several crop plants, including rice. Notably, strains ofPantoea ananatisandPantoea stewartiihave been found to cause bacterial leaf blight of rice in Togo and Benin, while other authors have observed thatPantoea agglomeranscan also cause bacterial leaf blight of rice. The contribution of these and perhaps other species ofPantoeato plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools.ResultUsing 34 whole genome sequences of the three-major plant-pathogenicPantoeaspecies, a set of PCR primers that specifically detect each of the three species,P. agglomerans,P. ananatis, andP. stewartii, was designed. A multiplex PCR protocol which can distinguish these three species and also detects members of otherPantoeaspecies was further developed. Upon validation on a set of reference strains, 609 suspectedPantoeastrains that were isolated from rice leaves or seeds originating from 11 African countries were screened. In total, 41P. agglomeransstrains from eight countries, 79P. ananatisstrains from nine countries, 269P. stewartiistrains from nine countries and 220 unsolvedPantoeastrains from ten countries were identified. The PCR protocol allowed detectingPantoeabacteria grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 5 ng/mL of total genomic DNA and 1 × 105CFU/mL of heated cells.ConclusionThis new molecular diagnostic tool will help accurately diagnose major plant-pathogenic species ofPantoea. Due to its robustness, specificity, sensitivity, and cost efficiency it will be very useful for plant protection services and for the epidemiological surveillance of these important crop-threatening bacteria.

Genomics-informed multiplex PCR scheme for rapid identification of rice-associated bacteria of the genus Pantoea

Plant Disease ◽  
2021 ◽  
Author(s):  
Kossi Kini ◽  
Raoul Agnimonhan ◽  
Rachelle Dossa ◽  
Drissa Silué ◽  
Ralf Koebnik
Keyword(s):  
Multiplex Pcr ◽  
Plant Protection ◽  
Detection Threshold ◽  
Leaf Blight ◽  
Crop Plants ◽  
Plant Diseases ◽  
Bacterial Leaf Blight ◽  
Epidemiological Surveillance ◽  
Molecular Diagnostic ◽  
Diagnostic Tools

The genus Pantoea forms a complex of more than 25 species, among which several cause diseases of various crop plants, including rice. Notably, strains of Pantoea ananatis and Pantoea stewartii have been repeatedly reported to cause bacterial leaf blight of rice, whereas other authors have observed that Pantoea agglomerans can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools. Using 32 whole genome sequences of the three major plant-pathogenic Pantoea species, a set of PCR primers that detect each of the three species, P. agglomerans, P. ananatis, and P. stewartii, was designed. A multiplex PCR scheme which can distinguish these three species and also detects members of other Pantoea species was further developed. Upon validation on a set of reference strains, 607 suspected Pantoea strains that were isolated from rice leaves or seeds originating from eleven African countries were screened. In total, 41 P. agglomerans strains from eight countries, 79 P. ananatis strains from nine countries, 269 P. stewartii strains from nine countries and 218 unresolved Pantoea strains from ten countries were identified. The PCR protocol allowed detecting Pantoea bacteria grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 0.5 ng/μl of total genomic DNA and 1×10^4 CFU/ml of heated cells. This new molecular diagnostic tool will help accurately diagnosing major plant-pathogenic species of Pantoea. Due to its robustness, specificity, sensitivity and cost efficiency, it will be very useful for plant protection services and for the epidemiological surveillance of these important crop-threatening bacteria.

scholarly journals A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

2019 ◽  
Author(s):  
Abdul Wahab ◽  
Ayaz Shaukat ◽  
Qasim Ali ◽  
Mubashir Hussain ◽  
Taj Ali Khan ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Disease Transmission ◽  
Diagnostic Performance ◽  
Detection Threshold ◽  
Plasmodium Species ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Diagnostic Tools ◽  
Sequencing Method

AbstractVarious PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput ‘haemoprotobiome’ metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

scholarly journals PCR and qPCR-based applications in rumen microbiology research: a review

2019 ◽  
Vol 23 (1) ◽  
Author(s):  
Isaac Malgwi ◽  
János Tossenberger ◽  
Veronika Halas ◽  
György Végvári ◽  
Melinda Kovács ◽  
...  
Keyword(s):  
Amplicon Sequencing ◽  
Microbial Genome ◽  
Metagenomic Data ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Complex Nature ◽  
Data Generation ◽  
Rumen Microbiota ◽  
Rumen Microbiome ◽  
Functional Characterisation

The rumen and its microbial ecosystem play a central role in the overall nutrition and health of ruminant animals. However, development and homeostatic state of the entire gut system is influenced by different interrelated factors. Recent developments in molecular diagnostic tools by using amplicon sequencing of 16S ribosomal RNA and use of high-throughput data generated through applications of pyrosequencing is a promising approach to defining the rumen microbial genome. Several procedures such as genome-wide shotgun sequencing for metagenomic data generation to predict how the rumen microbiota works, bacterial DNA integration in order to construct or edit genomes of isolated microbes and several other “omic”-based technologies based on PCR and real-time PCR (qPCR), have elucidated the complexity of the rumen microbiota. These tools are more sensitive and precise in quantitation, identification and functional characterisation of the entire rumen microbiome. PCR/qPCR enables investigations of changes in the microbiome and microbiota with respect to age, diet, species and environmental variations thus providing new information about rumen microbial genome. In this review, we will highlight recent findings using PCR and qPCR-based procedures in investigating the complex nature of the rumen microbial population which has advanced our knowledge and understanding of the rumen microbiome.

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 The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
James M. Hodge ◽  
Andrey A. Yurchenko ◽  
Dmitriy A. Karagodin ◽  
Reem A. Masri ◽  
Ryan C. Smith ◽  
...  
Keyword(s):  
Malaria Vector ◽  
Internal Transcribed Spacer ◽  
Single Species ◽  
Its2 Sequence ◽  
Pathogen Transmission ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Sequence Length ◽  
Phylogenetic Position ◽  
Internal Transcribed Spacer 2

Abstract Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

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.

scholarly journals LAMP in Neglected Tropical Diseases: A Focus on Parasites

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 521
Author(s):  
Juan García-Bernalt Diego ◽  
Pedro Fernández-Soto ◽  
Antonio Muro
Keyword(s):  
Neglected Tropical Diseases ◽  
Treatment Success ◽  
Public Health Problem ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
World Population ◽  
Major Public Health Problem ◽  
Tropical Diseases ◽  
Early 21St Century

Neglected Tropical Diseases (NTDs), particularly those caused by parasites, remain a major Public Health problem in tropical and subtropical regions, with 10% of the world population being infected. Their management and control have been traditionally hampered, among other factors, by the difficulty to deploy rapid, specific, and affordable diagnostic tools in low resource settings. This is especially true for complex PCR-based methods. Isothermal nucleic acid amplification techniques, particularly loop-mediated isothermal amplification (LAMP), appeared in the early 21st century as an alternative to PCR, allowing for a much more affordable molecular diagnostic. Here, we present the status of LAMP assays development in parasite-caused NTDs. We address the progress made in different research applications of the technique: xenomonitoring, epidemiological studies, work in animal models and clinical application both for diagnosis and evaluation of treatment success. Finally, we try to shed a light on the improvements needed to achieve a true point-of-care test and the future perspectives in this field.

scholarly journals A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Aušra Domanska ◽  
Justin W. Flatt ◽  
Joonas J. J. Jukonen ◽  
James A. Geraets ◽  
Sarah J. Butcher
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
High Resolution ◽  
Cultured Cells ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Parechovirus ◽  
Cryo Electron Microscopy

ABSTRACTHuman parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCEHuman parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

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