scholarly journals A Lab-on-a-Chip Device Integrated DNA Extraction and Solid Phase PCR Array for the Genotyping of High-Risk HPV in Clinical Samples

Micromachines ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 537 ◽  
Author(s):  
Cancan Zhu ◽  
Anzhong Hu ◽  
Junsheng Cui ◽  
Ke Yang ◽  
Xinchao Zhu ◽  
...  
Keyword(s):  
High Risk ◽  
Dna Extraction ◽  
Molecular Diagnostics ◽  
Solid Phase ◽  
Lab On A Chip ◽  
Clinical Diagnostics ◽  
Oligonucleotide Array ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Multiplex Amplification

Point-of-care (POC) molecular diagnostics play a crucial role in the prevention and treatment of infectious diseases. It is necessary to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. In this study, we proposed a lab-on-a-chip device that integrated DNA extraction, solid-phase PCR and genotyping detection. The ingenious design of the pneumatic microvalves enabled the fluid mixing and reagent storage to be organically combined, significantly reducing the size of the chip. The solid oligonucleotide array incorporated into the chip allowed the spatial separation of the primers and minimized undesirable interactions in multiplex amplification. As a proof-of-concept for POC molecular diagnostics on the device, five genotypes of high-risk human papillomavirus (HPV) (HPV16/HPV18/HPV31/HPV33/HPV58) were examined. Positive quality control samples and HPV patient cervical swab specimens were analyzed on the integrated microdevice. The platform was capable of detection approximately 50 copies of HPV virus per reaction during a single step, including DNA extraction, solid-phase PCR and genotype detection, in 1 h from samples being added to the chip. This simple and inexpensive microdevice provided great utility for the screening and monitoring of HPV genotypes. The sample-to-result platform will pave the way for wider application of POC molecular testing in the fields of clinical diagnostics, food safety, and environmental monitoring.

scholarly journals Are Staphylococcus intermedius Infections in Humans Cases of Mistaken Identity? A Case Series and Literature Review

2015 ◽  
Vol 2 (3) ◽  
Author(s):  
Roberto Viau ◽  
Andrea M. Hujer ◽  
Kristine M. Hujer ◽  
Robert A. Bonomo ◽  
Robin L.P. Jump
Keyword(s):  
Literature Review ◽  
Molecular Diagnostics ◽  
Case Series ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Pathogen ◽  
Detailed Review ◽  
Mistaken Identity ◽  
Staphylococcus Intermedius ◽  
Microbiological Methods

Abstract Staphylococcus intermedius and Staphylococcus pseudintermedius are difficult to distinguish using conventional microbiological methods. Molecular diagnostic tools change our understanding of the epidemiology of these 2 organisms. In this study, we present (1) a detailed review of the current literature on molecular diagnostics and (2) a case series in which misidentification was proven in 1 case. We conclude that S pseudintermedius is a more common human pathogen than previously recognized.

scholarly journals CRISPR-Cas Systems for Diagnosing Infectious Diseases

2020 ◽  
Author(s):  
Anastasiya Kostyusheva ◽  
Sergey Brezgin ◽  
Yurii Babin ◽  
Irina Vasil'eva ◽  
Dmitry Kostyushev ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Molecular Diagnostics ◽  
Large Scale ◽  
Point Of Care ◽  
Disease Spread ◽  
Detection Methods ◽  
Epidemiological Surveillance ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Wide Range

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas9, Cas12, Cas13, Cas14) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, or lateral flow assay detection. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing coronoviral nCov-2019 infection) urgently need the developing of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

scholarly journals Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

2016 ◽  
Vol 2 ◽  
Author(s):  
CORRADO MINETTI ◽  
E. JAMES LACOURSE ◽  
LISA REIMER ◽  
J. RUSSELL STOTHARD
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostics ◽  
Neglected Tropical Diseases ◽  
Monitoring And Evaluation ◽  
Diagnostic Methods ◽  
Future Application ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Future Research ◽  
Preventive Chemotherapy

SUMMARY The current mainstay for control of the four major helminth diseases in humans (lymphatic filariasis, onchocerciasis, soil-transmitted helminthiases and schistosomiasis) is with preventive chemotherapy by mass administration of key anthelminthics. Following the London Declaration on Neglected Tropical Diseases in 2012, a roadmap for the elimination and control of these helminthiases by 2020 has been devised. With expected declines in prevalence and intensity of these infections, there is urgent need for implementing more sensitive, high-throughput and cost-effective diagnostic tools. Currently available diagnostic approaches for surveying, monitoring and evaluating helminth control programmes are based on microscopical observation of eggs/larvae, and/or detection of antibodies or parasite antigens in stool, urine or blood; all relatively low-throughput and of limited sensitivity and specificity. Newly proposed approaches for helminthiases diagnosis include the nucleic acid-based methods of (multiplex) real-time polymerase chain reaction assays, loop-mediated isothermal amplification and recombinase polymerase amplification. However, as well as sensitivity/specificity evaluation, their comparison to current ‘gold standard’ diagnostics and future application in individual-/community-based diagnosis, or in xenomonitoring requires consideration of relative costs, agreement of standard methods and strategic interpretation of resulting data before control/elimination programmes might best utilize molecular diagnostics to inform decision making. We review current nucleic-acid-based molecular diagnostic methods and highlight the needs and future research required to refine these tools for monitoring and evaluation of control and elimination programmes for four major human helminthiases.

scholarly journals Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Roland Martzy ◽  
Katharina Bica-Schröder ◽  
Ádám Márk Pálvölgyi ◽  
Claudia Kolm ◽  
Stefan Jakwerth ◽  
...  
Keyword(s):  
Ionic Liquids ◽  
Dna Extraction ◽  
Bacterial Species ◽  
Model Organism ◽  
Reference Method ◽  
Clinical Diagnostics ◽  
Molecular Diagnostic ◽  
Bacterial Cells ◽  
Gram Positive ◽  
Rapid Preparation

Abstract The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

scholarly journals Applications of Nanobiotechnology in Clinical Diagnostics

2007 ◽  
Vol 53 (11) ◽  
pp. 2002-2009 ◽  
Author(s):  
Kewal K Jain
Keyword(s):  
Clinical Diagnosis ◽  
Molecular Diagnostics ◽  
Biomarker Discovery ◽  
Point Of Care ◽  
Clinical Diagnostics ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Commercial Development ◽  
Diagnostic Applications

Abstract Background: Nanobiotechnologies are being applied to molecular diagnostics and several technologies are in development. Methods: This review describes nanobiotechnologies that are already incorporated in molecular diagnostics or have potential applications in clinical diagnosis. Selected promising technologies from published literature as well as some technologies that are in commercial development but have not been reported are included. Results: Nanotechnologies enable diagnosis at the single-cell and molecule levels, and some can be incorporated in current molecular diagnostic methods, such as biochips. Nanoparticles, such as gold nanoparticles and quantum dots, are the most widely used, but various other nanotechnological devices for manipulation at the nanoscale as well as nanobiosensors are also promising for potential clinical applications. Conclusions: Nanotechnologies will extend the limits of current molecular diagnostics and enable point-of-care diagnostics, integration of diagnostics with therapeutics, and development of personalized medicine. Although the potential diagnostic applications are unlimited, the most important current applications are foreseen in the areas of biomarker discovery, cancer diagnosis, and detection of infectious microorganisms. Safety studies are needed for in vivo use. Because of its close interrelationships with other technologies, nanobiotechnology in clinical diagnosis will play an important role in the development of nanomedicine in the future.

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 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.

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

2018 ◽  
Vol 3 (5) ◽  
pp. e001069 ◽  
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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