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

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.

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

10.1101/580183 ◽

2019 ◽

Cited By ~ 1

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.

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Algorithmic and Stochastic Representations of Gene Regulatory Networks and Protein-Protein Interactions

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190311125256 ◽

2019 ◽

Vol 19 (6) ◽

pp. 413-425 ◽

Cited By ~ 3

Author(s):

Athanasios Alexiou ◽

Stylianos Chatzichronis ◽

Asma Perveen ◽

Abdul Hafeez ◽

Ghulam Md. Ashraf

Keyword(s):

Protein Interactions ◽

Biological Networks ◽

Regulatory Networks ◽

Review Paper ◽

Boolean Networks ◽

Diagnostic Tools ◽

Complex Nature ◽

Cellular Interactions ◽

Protein Protein Interactions ◽

Deterministic Models

Background:Latest studies reveal the importance of Protein-Protein interactions on physiologic functions and biological structures. Several stochastic and algorithmic methods have been published until now, for the modeling of the complex nature of the biological systems.Objective:Biological Networks computational modeling is still a challenging task. The formulation of the complex cellular interactions is a research field of great interest. In this review paper, several computational methods for the modeling of GRN and PPI are presented analytically.Methods:Several well-known GRN and PPI models are presented and discussed in this review study such as: Graphs representation, Boolean Networks, Generalized Logical Networks, Bayesian Networks, Relevance Networks, Graphical Gaussian models, Weight Matrices, Reverse Engineering Approach, Evolutionary Algorithms, Forward Modeling Approach, Deterministic models, Static models, Hybrid models, Stochastic models, Petri Nets, BioAmbients calculus and Differential Equations.Results:GRN and PPI methods have been already applied in various clinical processes with potential positive results, establishing promising diagnostic tools.Conclusion:In literature many stochastic algorithms are focused in the simulation, analysis and visualization of the various biological networks and their dynamics interactions, which are referred and described in depth in this review paper.

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Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan

Scientific Reports ◽

10.1038/s41598-021-91615-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kazutoshi Yoshitake ◽

Gaku Kimura ◽

Tomoko Sakami ◽

Tsuyoshi Watanabe ◽

Yukiko Taniuchi ◽

...

Keyword(s):

Time Series ◽

Microbial Communities ◽

Pacific Coast ◽

Three Dimensional ◽

Amplicon Sequencing ◽

Metagenomic Data ◽

Shotgun Metagenomics ◽

Functional Features ◽

Pacific Coast Of Japan ◽

Marine Microbial Communities

AbstractAlthough numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data (http://marine-meta.healthscience.sci.waseda.ac.jp/omd/), which provides a three-dimensional bird’s-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

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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 ◽

10.1007/s13205-020-02602-w ◽

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.

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

Malaria Journal ◽

10.1186/s12936-021-03676-4 ◽

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.

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LAMP in Neglected Tropical Diseases: A Focus on Parasites

Diagnostics ◽

10.3390/diagnostics11030521 ◽

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.

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The Microbiome Modeling Toolbox: from microbial interactions to personalized microbial communities

Bioinformatics ◽

10.1093/bioinformatics/bty941 ◽

2018 ◽

Vol 35 (13) ◽

pp. 2332-2334 ◽

Cited By ~ 16

Author(s):

Federico Baldini ◽

Almut Heinken ◽

Laurent Heirendt ◽

Stefania Magnusdottir ◽

Ronan M T Fleming ◽

...

Keyword(s):

Microbial Communities ◽

Microbial Interactions ◽

Microbial Genome ◽

Metagenomic Data ◽

Metabolic Interactions ◽

Constraint Based Modeling ◽

Genome Scale

Abstract Motivation The application of constraint-based modeling to functionally analyze metagenomic data has been limited so far, partially due to the absence of suitable toolboxes. Results To address this gap, we created a comprehensive toolbox to model (i) microbe–microbe and host–microbe metabolic interactions, and (ii) microbial communities using microbial genome-scale metabolic reconstructions and metagenomic data. The Microbiome Modeling Toolbox extends the functionality of the constraint-based reconstruction and analysis toolbox. Availability and implementation The Microbiome Modeling Toolbox and the tutorials at https://git.io/microbiomeModelingToolbox.

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

Journal of Virology ◽

10.1128/jvi.01597-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 5

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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The burden of congenital Chagas disease and implementation of molecular diagnostic tools in Latin America

BMJ Global Health ◽

10.1136/bmjgh-2018-001069 ◽

2018 ◽

Vol 3 (5) ◽

pp. e001069 ◽

Cited By ~ 21

Author(s):

Albert Picado ◽

Israel Cruz ◽

Maël Redard-Jacot ◽

Alejandro G Schijman ◽

Faustino Torrico ◽

...

Keyword(s):

Latin America ◽

Chagas Disease ◽

Diagnostic Algorithm ◽

Diagnostic Methods ◽

Diagnosis And Treatment ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Molecular Tests ◽

Epidemiological Impact ◽

And Performance

It is estimated that between 8000 and 15 000 Trypanosoma cruzi infected babies are born every year to infected mothers in Chagas disease endemic countries. Currently, poor access to and performance of the current diagnostic algorithm, based on microscopy at birth and serology at 8–12 months after delivery, is one of the barriers to congenital Chagas disease (CCD) control. Detection of parasite DNA using molecular diagnostic tools could be an alternative or complement to current diagnostic methods, but its implementation in endemic regions remains limited. Prompt diagnosis and treatment of CCD cases would have a positive clinical and epidemiological impact. In this paper, we analysed the burden of CCD in Latin America, and the potential use of molecular tests to improve access to early diagnosis and treatment of T. cruzi infected newborns.

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Frequency of benzimidazole resistance inHaemonchus contortus populations isolated from buffalo, goat and sheep herds

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612013000400015 ◽

2013 ◽

Vol 22 (4) ◽

pp. 548-553 ◽

Cited By ~ 12

Author(s):

Ronaldo Luiz Nunes ◽

Livia Loiola dos Santos ◽

Eduardo Bastianetto ◽

Denise Aparecida Andrade de Oliveira ◽

Bruno Santos Alves Figueiredo Brasil

Keyword(s):

Haemonchus Contortus ◽

Significant Variation ◽

Genetic Basis ◽

Anthelmintic Resistance ◽

Allelic Frequency ◽

Molecular Diagnostic ◽

Economic Losses ◽

Diagnostic Tools ◽

Ruminant Species ◽

Benzimidazole Resistance

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

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