Modern Approach to Medical Diagnostics - the Use of Separation Techniques in Microorganisms Detection

2019 ◽  
Vol 26 (1) ◽  
pp. 121-165 ◽  
Author(s):  
Agnieszka Chylewska ◽  
Małgorzata Ogryzek ◽  
Mariusz Makowski
Keyword(s):  
Nucleic Acid ◽  
Pathogen Detection ◽  
Medical Diagnostics ◽  
Detection Methods ◽  
Immunological Methods ◽  
Separation Techniques ◽  
Modern Approach ◽  
Food Protection ◽  
Wide Range

Background:Analytical chemistry and biotechnology as an interdisciplinary fields of science have been developed during many years and are experiencing significant growth, to cover a wide range of microorganisms separation techniques and methods, utilized for medical therapeutic and diagnostic purposes. Currently scientific reports contribute by introducing electrophoretical and immunological methods and formation of devices applied in food protection (avoiding epidemiological diseases) and healthcare (safety ensuring in hospitals).Methods:Electrophoretic as well as nucleic-acid-based or specific immunological methods have contributed tremendously to the advance of analyses in recent three decades, particularly in relation to bacteria, viruses and fungi identifications, especially in medical in vitro diagnostics, as well as in environmental or food protection.Results:The paper presents the pathogen detection competitiveness of these methods against conventional ones, which are still too time consuming and also labor intensive. The review is presented in several parts following the current trends in improved pathogens separation and detection methods and their subsequent use in medical diagnosis.Discussion:Part one, consists of elemental knowledge about microorganisms as an introduction to their characterization: descriptions of divisions, sizes, membranes (cells) components. Second section includes the development, new technological and practical solution descriptions used in electrophoretical procedures during microbes analyses, with special attention paid to bio-samples analyses like blood, urine, lymph or wastewater. Third part covers biomolecular areas that have created a basis needed to identify the progress, limitations and challenges of nucleic-acid-based and immunological techniques discussed to emphasize the advantages of new separative techniques in selective fractionating of microorganisms.

scholarly journals Rapid detection methods of microbial pathogens in foods – a short survey

2021 ◽  
Vol 8 (1) ◽  
pp. 5-22
Author(s):  
Barbora Vidová ◽  
Andrej Godány ◽  
Ernest Šturdík
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Pathogen Detection ◽  
Microbial Pathogens ◽  
Detection Methods ◽  
Laboratory Methods ◽  
Microbial Pathogen ◽  
Short Survey ◽  
Wide Range ◽  
The World

During harvesting, processing and handling operations foods may become contaminated with a wide range of microorganisms. This paper is presented as a short survey of recent used laboratory methods for foods microbial pathogen detection, briefly summarizing rapid, specific and sensitive methods useful for foods testing based on immunochemical and nucleic acid technologies. As the world becomes more concerned with safe foods, the demand for rapid detecting will only increase.

scholarly journals SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

2020 ◽  
Author(s):  
Mujahed I. Mustafa ◽  
Abdelrafie M. Makhawi
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Emerging Infectious Diseases ◽  
Cost Effective ◽  
Detection Methods ◽  
Diagnostic Tools ◽  
Nucleic Acid Detection ◽  
Nucleic Acid Binding

Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases is a better understanding of its pathogenesis. According to WHO, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free and cost-effective. Nucleic acid pathogen detection methods, typically PCR have numerous limitations, such as highly sophisticated equipments, reagents, and trained personnel rely on well-established laboratories beside time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools with rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit on unpredicted in vitro properties CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic-acid binding event. These effectors are attached with a diversity of reporters and utilized in tandem with present of isothermal amplification approaches to create sensitive identification in multiple field deployable formats. Although still in their beginning, yet SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious disease, with ultra-sensitive tests that don't require a lot of complicated processing. This review described SHERLOCK and DETECTR technologies beside their properties, functions, and perspectives to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

scholarly journals Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification

2004 ◽  
Vol 70 (8) ◽  
pp. 4727-4732 ◽  
Author(s):  
Erica T. Casper ◽  
John H. Paul ◽  
Matthew C. Smith ◽  
Michael Gray
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Wildlife Conservation ◽  
Red Tide ◽  
Diagnostic Testing ◽  
Karenia Brevis ◽  
Nucleic Acid Sequence ◽  
Cell Counts ◽  
Wide Range

ABSTRACT Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

scholarly journals Combining Inulin Multifunctional Polycation and Magnetic Nanoparticles: Redox-Responsive siRNA-Loaded Systems for Magnetofection

Polymers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 889 ◽  
Author(s):  
Carla Sardo ◽  
Emanuela Fabiola Craparo ◽  
Barbara Porsio ◽  
Gaetano Giammona ◽  
Gennara Cavallaro
Keyword(s):  
Magnetic Field ◽  
Nucleic Acid ◽  
Sirna Delivery ◽  
Superparamagnetic Iron Oxide ◽  
Cell Uptake ◽  
Nucleic Acid Delivery ◽  
Crosslinking Process ◽  
Wide Range ◽  
Theranostic Applications

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) are recognized as one of the most promising agents for theranostic applications. Among methods designed for siRNA delivery, magnetofection, that is, nucleic acid cell uptake under the influence of a magnetic field acting on magnetic nucleic acid vectors, is emerging as a unique approach to combining advantages such as strong improvement of the kinetics of the delivery process and the possibility of localizing nucleic acid delivery to an area where the magnetic field is applied. This paper reports on the preparation of siRNA loaded magnetoplexes—named ICD@SS@SPIONs/siRNA—by controlled crosslinking, in the presence of SPIONs, of the polycation INU-C-DETA, synthesized starting from the polysaccharide inulin by grafting diethylenetriamine and cystamine molecules. The obtained ICD@SS@SPIONs/siRNA have suitable chemical-physical characteristics to be employed for iv administration and are also able to release siRNA in a redox-triggered manner thanks to intracellular glutathione (GSH) mediated reduction of disulphide bridges formed during the crosslinking process. Moreover, ICD@SS@SPIONs/siRNA are able to produce magnetic targeting in vitro on breast cancer cells, without appreciable cyto- and hemo-toxic effects, in a wide range of concentrations. Finally, protein binding to nanoparticles revealed that obtained systems are potentially longer circulating and applicable as a smart multifunctional agents for cancer therapy.

scholarly journals Functionalized nanomaterials: their use as contrast agents in bioimaging: mono- and multimodal approaches

2013 ◽  
Vol 2 (2) ◽  
pp. 125-169 ◽  
Author(s):  
Quentin Le Trequesser ◽  
Hervé Seznec ◽  
Marie-Hélène Delville
Keyword(s):  
Contrast Agents ◽  
Photoacoustic Imaging ◽  
Imaging Techniques ◽  
Ion Beam ◽  
Medical Diagnostics ◽  
Molecular Probes ◽  
The Body ◽  
Wide Range

AbstractThe successful development of nanomaterials illustrates the considerable interest in the development of new molecular probes for medical diagnosis and imaging. Substantial progress was made in the synthesis protocol and characterization of these materials, whereas toxicological issues are sometimes incomplete. Nanoparticle-based contrast agents (CAs) tend to become efficient tools for enhancing medical diagnostics and surgery for a wide range of imaging modalities. The multimodal nanoparticles (NPs) are much more efficient than the conventional molecular-scale CAs. They provide new abilities for in vivo detection and enhanced targeting efficiencies through longer circulation times, designed clearance pathways, and multiple binding capacities. Properly protected, they can safely be used for the fabrication of various functional systems with targeting properties, reduced toxicity, and proper removal from the body. This review mainly describes the advances in the development of mono- to multimodal NPs and their in vitro and in vivo relevant biomedical applications ranging from imaging and tracking to cancer treatment. Besides the specific applications for classical imaging (magnetic resonance imaging, positron emission tomography, computed tomography, ultrasound, and photoacoustic imaging), the less common imaging techniques such as terahertz molecular imaging (THMI) or ion beam analysis (IBA) are mentioned. The perspectives on the multimodal theranostic NPs and their potential for clinical advances are also mentioned.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Virus Detection: A Review of the Current and Emerging Molecular and Immunological Methods

2021 ◽  
Vol 8 ◽  
Author(s):  
A. Cassedy ◽  
A. Parle-McDermott ◽  
R. O’Kennedy
Keyword(s):  
Nucleic Acid ◽  
Virus Detection ◽  
High Sensitivity ◽  
High Volume ◽  
Detection Methods ◽  
Immunological Methods ◽  
Nucleic Acid Detection ◽  
Viral Genomes ◽  
Diagnostic Measures ◽  
Point Of Use

Viruses are ubiquitous in the environment. While many impart no deleterious effects on their hosts, several are major pathogens. This risk of pathogenicity, alongside the fact that many viruses can rapidly mutate highlights the need for suitable, rapid diagnostic measures. This review provides a critical analysis of widely used methods and examines their advantages and limitations. Currently, nucleic-acid detection and immunoassay methods are among the most popular means for quickly identifying viral infection directly from source. Nucleic acid-based detection generally offers high sensitivity, but can be time-consuming, costly, and require trained staff. The use of isothermal-based amplification systems for detection could aid in the reduction of results turnaround and equipment-associated costs, making them appealing for point-of-use applications, or when high volume/fast turnaround testing is required. Alternatively, immunoassays offer robustness and reduced costs. Furthermore, some immunoassay formats, such as those using lateral-flow technology, can generate results very rapidly. However, immunoassays typically cannot achieve comparable sensitivity to nucleic acid-based detection methods. Alongside these methods, the application of next-generation sequencing can provide highly specific results. In addition, the ability to sequence large numbers of viral genomes would provide researchers with enhanced information and assist in tracing infections.

scholarly journals A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

2021 ◽  
Author(s):  
Ekaterina Kropocheva ◽  
Anton Kuzmenko ◽  
Alexei A. Aravin ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
Bacterial Cells ◽  
Dna And Rna ◽  
Rna Targets ◽  
Wide Range ◽  
Mesophilic Bacterium ◽  
Strict Specificity ◽  
Programmable Nucleases

ABSTRACTArgonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

scholarly journals An appraisal of various pathogen detection methods in eggs and poultry

2020 ◽  
Vol 1 (2) ◽  
pp. 93-97
Author(s):  
A. A. P. Milton ◽  
G. Bhuvana Priya ◽  
K. M. Momin ◽  
M. Angappan ◽  
Samir Das ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
False Negative ◽  
Foodborne Pathogen ◽  
Detection Methods ◽  
Immunological Methods ◽  
Culturable Bacteria ◽  
Product Recalls ◽  
Poultry Products ◽  
Negative Results ◽  
False Negative Results

Abstract: To ensure safety in egg and poultry products, timely detection of pathogenic microbes is of paramount importance. This review offers an appraisal of different routinely used and novel emerging pathogen detection methods in egg, poultry and their products. Timely detection of pathogens is decisive to curtail outbreak risks, reduce hospitalisation, and provide product assurance. It will also reduce the cost of holding food products in cold storage and reduces product recalls. Some crucial issues need to be taken care of in choosing or developing a foodborne pathogen detection method. They are requirement of costly or sophisticated equipment, portability, trained personnel, viable but non-culturable bacteria (may give false-negative results), dead microbes (may give false-positive results), stressed or sub-lethally damaged bacteria and slow-growing microbes (require enrichment). In this review, the focus has been given on culture-based methods, nucleic acid-based methods, immunological methods and biosensor based methods. Keywords: Egg; poultry; detection methods; product assurance; safety.

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