Electrophoretic approaches to sample collection and preparation for nucleic acids analysis

2014 ◽  
pp. 355-369
Author(s):  
C. Bradburne
Keyword(s):  
Nucleic Acids ◽  
Sample Collection

scholarly journals DETEKSI DINI DEMAM BERDARAH DENGUE DENGAN PEMERIKSAAN ANTIGEN NS1

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Mayer F. Wowor
Keyword(s):  
Nucleic Acids ◽  
Acute Phase ◽  
Vaccine Development ◽  
Early Stage ◽  
Serological Test ◽  
Clinical Care ◽  
Dengue Infection ◽  
Diagnostic Methods ◽  
Sample Collection ◽  
Ns1 Antigen

Abstract: Efficient and accurate diagnosis of dengue is of primary importance for clinical care, surveillance activities, outbreak control, pathogenesis, academic research, vaccine development, and clinical trials. Laboratory diagnostic methods for confirming dengue virus infection may involve detection of the viruses, viral nucleic acids, antigens and antibodies, or a combination of these techniques. After the onset of illness, the virus can be detected in serum, plasma, circulating blood cells, and other tissues for 4–5 days. During the early stage of the disease, virus isolation and the detection of viral nucleic acids or antigens, can be used to confirm the diagnosis of dengue infection. NS1 antigen appears as early as day 1 after the onset of fever, and declines to undetectable levels after day 5–6. At the end of the acute phase of infection, a serological test is the method of choice for diagnosis. A range of laboratory diagnostic methods has been developed to support patient management and disease control. The choice of diagnostic method depends on the purpose for which the test is done, available  laboratory facilities and technical expertise, costs, and the time of sample collection. Keywords: laboratory diagnostic methods, acute phase, NS1 antigen     Abstrak: Diagnosis dengue yang efisien dan akurat adalah hal yang paling penting  untuk proses perawatan di klinik, aktivitas surveilens, kontrol penularan penyakit, patogenesis, penelitian akademik, pengembangan vaksin dan percobaan-percobaan klinis. Metode diagnosis laboratorium untuk mengonfirmasi adanya infeksi virus dengue dapat meliputi deteksi virus dengue, asam nukleat virus, antigen dan antibodi, atau kombinasi dari teknik-teknik tersebut. Setelah serangan penyakit, virus dapat dideteksi dalam serum, plasma, sel-sel darah yang bersirkulasi, dan di jaringan lain dalam waktu 4-5 hari. Selama tahap awal dari penyakit, isolasi virus, asam nukleat virus, atau deteksi antigen dapat digunakan untuk diagnosis infeksi. Antigen NS1 muncul pada hari pertama setelah serangan demam dan menurun ke tingkat tidak terdeteksi setelah 5-6 hari. Pada akhir fase akut infeksi, serologi adalah metode pilihan untuk diagnosis. Metode diagnosis laboratorium telah berkembang untuk menunjang penanganan pasien dan kontrol penyakit. Pilihan untuk metode diagnosis bergantung pada tujuan tes dilakukan, fasilitas laboratorium dan tenaga ahli yang tersedia, biaya, dan saat sampel dikumpulkan. Kata kunci: metode diagnosis laboratorium, fase akut, antigen NS1

scholarly journals Plasmodium falciparum Allelic Diversity: A Comparison of DNA Extraction from Isolates Collected on Rapid Diagnostic Tests (Rdts) and Filter Paper

2021 ◽  
Author(s):  
Denise Patricia Mawili-Mboumba ◽  
Marie Louise Tshibola Mbuyi ◽  
Noe Patrick M’bondoukwe ◽  
Marielle Karine Bouyou-Akotet
Keyword(s):  
Plasmodium Falciparum ◽  
Nucleic Acids ◽  
Filter Paper ◽  
Diagnostic Tests ◽  
Allelic Diversity ◽  
Storage Conditions ◽  
Rapid Diagnostic Tests ◽  
Sample Collection ◽  
Filter Papers

Background: To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity. Methods: Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification. Results: Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs -20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples. Conclusion: These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.

scholarly journals BIOME-Preserve: A Novel Storage and Transport Medium for Preserving Anaerobic Microbiota Samples for Culture Recovery

2020 ◽  
Author(s):  
Embriette R. Hyde ◽  
Hiram Lozano ◽  
Steven Cox
Keyword(s):  
Nucleic Acids ◽  
Human Health ◽  
Anaerobic Bacteria ◽  
Human Microbiome ◽  
Sample Collection ◽  
Culture Collections ◽  
Oxygen Sensitive ◽  
The One ◽  
The Relationship ◽  
Human Stool

AbstractCulture-based study design is critical to advance research into the relationship between human health and the microbiome. Traditional sample collection protocols are focused on preserving nucleic acids and metabolites and are largely inappropriate for preserving sensitive anaerobic bacteria alive for later culture recovery. Here we introduce a novel microbiome preservation kit (BIOME-Preserve) that facilitates recovery of anaerobic organisms from human stool held at room temperature. Using a combination of culture recovery and shallow whole-genome shotgun sequencing, we characterized the culturable anaerobes from fresh human stool and from human stool held in BIOME-Preserve for up to 120 hours. We recovered several species of interest to microbiome researchers, including Bifidobacterium spp., Bacteroides spp., Blautia spp., Eubacterium halii, Akkermansia muciniphila, and Faecalibacterium prausnitzii. Together, our results suggest BIOME-Preserve is practical for the collection, transport, and culture of anaerobic bacteria from human samples and can help provide the foundation for culture collections that can be used in further research and in the development of microbiome-based therapeutics.ImportanceSequencing-based protocols for studying the human microbiome have unearthed a wealth of information about the relationship between the microbiome and human health. But these microbes cannot be leveraged as therapeutic targets without culture-based studies to phenotype species of interest and to establish culture collections for use in animal models. Contrary to popular opinion, most gastrointestinal bacteria can be cultured, yet most sample collection strategies are optimized for the preservation of nucleic acids and/or metabolites only and do not take into account considerations for preserving oxygen-sensitive anaerobes and facultative anaerobes, which comprise the majority of the human gut microbiome. A human microbiome sample transport and preservation medium such as the one described here can play an important role in enabling researchers to better understand the link between the microbiome and human health and how to leverage that link through novel microbiome-based therapeutics.

scholarly journals Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

2020 ◽  
Vol 21 (22) ◽  
pp. 8634
Author(s):  
Zuzana Pös ◽  
Ondrej Pös ◽  
Jakub Styk ◽  
Angelika Mocova ◽  
Lucia Strieskova ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Biomedical Applications ◽  
Clinical Care ◽  
Biological Properties ◽  
Sample Collection ◽  
Bacterial Dna ◽  
Sequence Composition ◽  
Different Types ◽  
Methodological Aspects ◽  
Informational Value

Analyzes of cell-free nucleic acids (cfNAs) have shown huge potential in many biomedical applications, gradually entering several fields of research and everyday clinical care. Many biological properties of cfNAs can be informative to gain deeper insights into the function of the organism, such as their different types (DNA, RNAs) and subtypes (gDNA, mtDNA, bacterial DNA, miRNAs, etc.), forms (naked or vesicle bound NAs), fragmentation profiles, sequence composition, epigenetic modifications, and many others. On the other hand, the workflows of their analyzes comprise many important steps, from sample collection, storage and transportation, through extraction and laboratory analysis, up to bioinformatic analyzes and statistical evaluations, where each of these steps has the potential to affect the outcome and informational value of the performed analyzes. There are, however, no universal or standard protocols on how to exactly proceed when analyzing different cfNAs for different applications, at least according to our best knowledge. We decided therefore to prepare an overview of the available literature and products commercialized for cfNAs processing, in an attempt to summarize the benefits and limitations of the currently available approaches, devices, consumables, and protocols, together with various factors influencing the workflow, its processes, and outcomes.

scholarly journals Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests

2020 ◽  
Author(s):  
Etienne A. Guirou ◽  
Tobias Schindler ◽  
Salome Hosch ◽  
Olivier Tresor Donfack ◽  
Charlene Aya Yoboue ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Diagnostic Tests ◽  
Large Scale ◽  
Rapid Diagnostic Tests ◽  
Nucleic Acid Amplification ◽  
Nucleotide Polymorphisms ◽  
Sample Collection ◽  
Malaria Surveillance ◽  
Bioko Island ◽  
Asymptomatic Patients

AbstractThe use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays

2008 ◽  
Vol 80 (9) ◽  
pp. 1684-1688 ◽  
Author(s):  
Reinhard B. Raggam ◽  
Jasmin Wagner ◽  
Birgit D. A. Michelin ◽  
Csilla Putz-Bankuti ◽  
Andreas Lackner ◽  
...  
Keyword(s):  
Liquid Phase ◽  
Nucleic Acids ◽  
Oral Fluid ◽  
Sample Collection ◽  
Molecular Assays ◽  
Reliable Detection

A review on salivary biomarkers in carcinoma diagnosis

2021 ◽  
Vol 1 (1) ◽  
pp. 013-021
Author(s):  
Meena Rakesh Kumar ◽  
Pabri Reena ◽  
Singh Prashant ◽  
Rani Priya ◽  
Raj Shobhit ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Salivary Glands ◽  
Rapid Detection ◽  
Invasive Carcinoma ◽  
Cost Effective ◽  
Minor Salivary Glands ◽  
Sample Collection ◽  
Non Invasive ◽  
Salivary Biomarkers ◽  
The U.S

Carcinoma is the 2nd leading mortality in the U.S. Signs & symptoms include typically unspecific until the tumours metastasize. Hence, an urgency is there for quick, precise, and non-invasive carcinoma diagnosis, rapid detection, diagnosis, stage surveys, & forecasts. Saliva is a multi-structural fluid, found in the oral region, containing secretions from primary and minor salivary glands. Species can even be found in blood-present molecules including Deoxyribo Nucleic Acids, RNAs, hormones, metabolites, and microbiota. Recently, saliva testing received considerable interest in identifying specific biomarkers as sample collection and processing is quick, cost-effective, accurate and doesn’t put any distress on the patient. We examine recent salivary biomarkers of systemic carcinoma by separating them into genomically, transcriptomically, proteomically, metabolomic and microbially dependent forms.

Electron Microscopy of Nucleic Acids

1974 ◽  
Vol 32 ◽  
pp. 302-303
Author(s):  
Norman Davidson
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acids ◽  
Basic Protein ◽  
Molecular Biologist ◽  
Topological Properties ◽  
Protein Film ◽  
Polynucleotide Chain

The basic protein film technique for mounting nucleic acids for electron microscopy has proven to be a general and powerful tool for the working molecular biologist in characterizing different nucleic acids. It i s possible to measure molecular lengths of duplex and single-stranded DNAs and RNAs. In particular, it is thus possible to as certain whether or not the nucleic acids extracted from a particular source are or are not homogeneous in length. The topological properties of the polynucleotide chain (linear or circular, relaxed or supercoiled circles, interlocked circles, etc. ) can also be as certained.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

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