A Rapid Nucleic Acid Detection Platform Based on Phosphorothioate-DNA and Sulfur Binding Domain
In: SYNBIO-D-22-00106
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In: SYNBIO-D-22-00106
SSRN
In: PNAS nexus, Band 1, Heft 1
ISSN: 2752-6542
Abstract
Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.
In: AACH-D-23-00038
SSRN
In: Public health genomics, Band 12, Heft 5-6, S. 308-318
ISSN: 1662-8063
Human papillomaviruses (HPV) are the etiologic agents of cancer of the uterine cervix and several other neoplasias. Detection of HPV infection will improve the sensitivity of primary and secondary screening of cervical cancer. The clinical indications for the use of HPV tests will have to consider the natural history of HPV infection and diseases, and the multiplicity of types involved. Signal amplification HPV DNA tests detect several high-risk HPV types, are standardized, commercially available and approved for clinical use. Nucleic acid amplification techniques are ideal methods for epidemiologic purposes since they minimize misclassification of HPV infection status and allow detection of infection with low viral burden. They are currently under evaluation for clinical use. PCR is the most widespread method for HPV typing, especially with the use of consensus primers and typing with reverse hybridization techniques. Novel promising HPV detection strategies are now proposed, such as HPV mRNA detection, and suspension or solid phase arrays. These novel techniques will have to be evaluated as stringently as actual assays in clinical studies. Although assays have been developed for the evaluation of viral load, viral integration and HPV polymorphism in molecular epidemiological studies, their role in clinical practice is not currently defined.
In: TRAC-D-21-00354
SSRN
In: Defence science journal: DSJ, Band 41, Heft 4, S. 335-356
ISSN: 0011-748X
The increasing availability and decreasing cost of custom synthetic nucleic acids presents risk of misuse. The authors provide recommendations to implement comprehensive screening and secure commercial nucleic acid synthesis services against misuse.
SWP
Salmonella is a leading source of bacterial foodborne illness in humans, causing gastroenteritis outbreaks with bacteraemia occurrences that can lead to clinical complications and death. Eggs, poultry and pig products are considered as the main carriers of the pathogenic Salmonella for humans. To prevent this relevant zoonosis, key changes in food safety regulations were undertaken to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis were reported worldwide in the last decade. Thus, new strategies for Salmonella detection are a priority for both, food safety and public health authorities. Such detection systems should provide significant reduction in diagnostic time (hours) compared to the currently available methods (days). Herein, we report on the discovery and characterization of nucleic acid probes for the sensitive and specific detection of live Salmonella within less than 8 h of incubation. We are the first to postulate the nuclease activity derived from Salmonella as biomarker of infection and its utility to develop innovative detection strategies. Our results have shown the screening and identification of two oligonucleotide sequences (substrates) as the most promising probes for detecting Salmonella - Sal-3 and Sal-5. The detection limits for both probes were determined with the reference Salmonella Typhimurium (STM 1) and Salmonella Enteritidis (SE 1) cultures. Sal-3 has reported LOD values around 10(5) CFU mL(-1) for STM 1 and 10(4) CFU mL(-1) for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 10(4) CFU mL(-1) for STM 1 and 10(4) CFU mL(-1) for SE 1. Both selected probes have shown the capability to recognize 49 out of 51 different Salmonella serotypes tested in vitro and the most frequent serotypes in porcine mesenteric lymph nodes as a standard sample used in fattening-pig salmonellosis baseline studies. Notably, our results showed 100% correlation between nuclease detection and the PCR-InvA or ISO-6579 standard method, underlining the great potential of this innovative nucleic acids technology to be implemented as a rapid method for food safety testing. (C) 2018 Elsevier B.V. All rights reserved. ; Funding Agencies|MINNECO [PTQ-16-08414]; Centre for the Development of Industrial Technology [CDTI] [20161256]; Departamento de Industria, Energia e Innovacion of the Navarra government, Spain [2016 PT071, 2017 PT031]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg foundation
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Abstract: The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy
Nucleic acid testing (NAT) played a crucial role in containing the spread of SARS-CoV-2 during the epidemic. The gold standard technique, the quantitative real-time polymerase chain reaction (qRT-PCR) technique, is currently used by the government and medical boards to detect SARS-CoV-2. Due to the limitations of this technology, it is not capable of meeting the needs of large-scale rapid detection. To solve this problem, many new techniques for detecting nucleic acids of SARS-CoV-2 have been reported. Therefore, a review that systematically and comprehensively introduces and compares various detection technologies is needed. In this paper, we not only review the traditional NAT but also provide an overview of microfluidic-based NAT technologies and summarize and discuss the characteristics and development prospects of these techniques.
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In: Environmental science and pollution research: ESPR, Band 30, Heft 4, S. 10346-10359
ISSN: 1614-7499
In: Springer eBook Collection
1 Introduction -- References -- 2 The structure of the nucleic acids -- 2.1 Monomeric components -- 2.1.1 Pyrimidine bases -- 2.1.2 Purine bases -- 2.1.3 Pentose and deoxypentose sugars -- 2.1.4 Nucleosides -- 2.1.5 Nucleotides -- 2.2 The primary structure of the nucleic acids -- 2.3 Shorthand notation -- 2.4 Base composition analysis of DNA -- 2.5 Molecular weight of DNA -- 2.6 The secondary structure of DNA -- 2.6.1 The basic structures -- 2.6.2 Variations on the B-form of DNA -- 2.6.3 Z-DNA -- 2.6.4 The dynamic structure of DNA -- 2.7 Denaturation and renaturation -- 2.7.1 DNA denaturation: the helix-coil transition -- 2.7.2 The renaturation of DNA: C0t value analysis -- 2.7.3 The buoyant density of DNA -- 2.8 Supercoils, cruciforms and triple-stranded structures -- 2.9 The secondary and tertiary structure of RNA -- 2.10 Chemical reactions of bases, nucleotides and polynucleotides -- 2.10.1 Reactions of ribose and deoxyribose -- 2.10.2 Reactions of the bases -- 2.10.3 Phosphodiester bond cleavage -- 2.10.4 Photochemistry -- References -- 3 Chromosome organization -- 3.1 Introduction -- 3.2 Eukaryote DNA -- 3.2.1 The eukaryote cell cycle -- 3.2.2 Eukaryote chromosomes -- 3.2.3 The allocation of specific genes to specific chromosomes -- 3.2.4 Haploid DNA content (C value) -- 3.2.5 Gene frequency -- 3.2.6 Eukaryote gene structure -- 3.3 Chromatin structure -- 3.3.1 Histones and non-histone proteins -- 3.3.2 The nucleosome -- 3.3.3 Nucleosome phasing -- 3.3.4 Higher orders of chromatin structure -- 3.3.5 Loops, matrix and the chromosome scaffold -- 3.3.6 Lampbrush chromosomes -- 3.3.7 Polytene chromosomes -- 3.4 Extranuclear DNA -- 3.4.1 Mitochondrial DNA -- 3.4.2 Chloroplast DNA -- 3.4.3 Kinetoplast DNA -- 3.5 Bacteria -- 3.5.1 The bacterial chromosome -- 3.5.2 The bacterial division cycle -- 3.5.3 Bacterial transformation -- 3.6 Viruses -- 3.6.1 Structure -- 3.6.2 Virus classification -- 3.6.3 Life cycle -- 3.6.4 The Hershey-Chase experiment -- 3.6.5 Virus mutants -- 3.6.6 Virus nucleic acids -- 3.6.7 The information content of viral nucleic acids -- 3.6.8 Lysogeny and transduction -- 3.6.9 Tumour viruses and animal cell transformation -- 3.6.10 Viroids -- 3.6.11 Prions -- 3.7 Plasmids and transposons 77 -- References -- 4 Degradation and modification of nucleic acids -- 4.1 Introduction and classification of nucleases -- 4.2 Non-specific nucleases -- 4.2.1 Non-specific endonucleases -- 4.2.2 Non-specific exonucleases -- 4.3 Ribonucleases (RNases) -- 4.3.1 Endonucleases which form 3?-phosphate groups -- 4.3.2 Endonucleases which form 5?-phosphate groups -- 4.3.3 RNA exonucleases -- 4.3.4 Ribonucleases which act on RNA:DNA hybrids (RNase H) -- 4.3.5 Double-stranded RNA-specific ribonucleases -- 4.3.6 Ribonuclease inhibitors -- 4.4 Polynucleotide phosphorylase (PNPase) -- 4.5 Deoxy ribonucleases (DNases) -- 4.5.1 Endonucleases -- 4.5.2 Exonucleases -- 4.5.3 Restriction endonucleases -- 4.6 Nucleic acid methylation -- 4.6.1 DNA methylation -- 4.6.2 RNA methylation and other RNA nucleotide modifications -- 4.7 Nucleic acid kinases and phosphatases -- 4.7.1 Bacteriophage polynucleotide kinase -- 4.7.2 Eukaryotic DNA and RNA kinases -- 4.8 Base exchange in RNA and DNA -- References -- 5 The metabolism of nucleotides -- 5.1 Anabolic pathways -- 5.2 The biosynthesis of the purines -- 5.3 Preformed purines as precursors -- 5.4 The biosynthesis of the pyrimidines -- 5.5 The biosynthesis of deoxyribonucleotides and its control -- 5.6 The biosynthesis of thymine derivatives -- 5.7 Aminopterin in selective media -- 5.8 Formation of nucleoside triphosphates -- 5.9 General aspects of catabolism -- 5.10 Purine catabolism -- 5.11 Pyrimidine catabolism -- References -- 6 Replication of DNA -- 6.1 Introduction -- 6.2 Semiconservative replication -- 6.3 The replication fork -- 6.3.1 Discontinuous synthesis -- 6.3.2 Okazaki pieces -- 6.3.3 Direction of chain growth -- 6.3.4 Initiation of Okazaki pieces -- 6.3.5 Continuous synthesis -- 6.4 Enzymes of DNA synthesis -- 6.4.1 Introduction -- 6.4.2 DNA polymerases -- 6.4.3 DNA ligases -- 6.4.4 Helix-destabilizing proteins (HD) or single-stranded DNA- binding proteins (ssb) -- 6.4.5 DNA unwinding proteins or DNA helicases (DNA-dependent ATPases) -- 6.4.6 Topoisomerases -- 6.5 Fidelity of replication -- 6.6 In vitro systems for studying DNA replication -- 6.6.1 dna mutants -- 6.6.2 Permeable cells -- 6.6.3 Cell lysates -- 6.6.4 Soluble extracts -- 6.6.5 Reconstruction experiments -- 6.7 Molecular biology of the replication fork -- 6.7.1 Lagging-strand synthesis -- 6.7.2 Leading-strand synthesis -- 6.7.3 RF replication -- 6.8 Initiation of replication-general -- 6.8.1 Methods of locating the origin and direction of replication -- 6.8.2 Replicons -- 6.8.3 Rate of replication -- 6.8.4 Origin strategies -- 6.8.5 Positive or negative control of initiation -- 6.9 Initiation of replication-specific examples -- 6.9.1 Small single-stranded phage -- 6.9.2 Double-stranded phage -- 6.9.3 Plasmids -- 6.9.4 Bacteria -- 6.9.5 Mitochondria -- 6.9.6 Double-stranded cyclic DNA viruses (SV40 and polyoma) -- 6.9.7 Adenoviruses -- 6.9.8 Yeast -- 6.9.9 Higher eukaryotes -- 6.9.10 Retroviruses -- 6.10 Termination of replication -- 6.10.1 Cyclic chromosomes -- 6.10.2 Small linear chromosomes -- 6.10.3 Telomeres -- 6.11 Replication complexes -- 6.12 Chromatin replication -- References -- 7 Repair, recombination and DNA rearrangement -- 7.1 Introduction -- 7.2 Mutations and mutagens -- 7.2.1 Base and nucleoside analogues -- 7.2.2 Alkylating agents -- 7.2.3 Intercalating agents -- 7.2.4 The effects of ionizing radiation -- 7.2.5 Ultraviolet radiation -- 7.3 Repair mechanisms -- 7.3.1 Reversal of damage -- 7.3.2 Excision repair -- 7.3.3 Mismatch repair -- 7.3.4 Post-replication repair -- 7.4 Recombination -- 7.4.1 E. coli rec system and single-strand invasion -- 7.4.2 Reciprocal recombination between duplex DNA molecules -- 7.4.3 Site-specific recombination -- 7.5 Gene amplification -- 7.5.1 Developmental amplification -- 7.5.2 Amplification by chemical selection -- 7.5.3 Mechanism of amplification -- 7.6 Gene duplication and pseudogenes -- 7.6.1 Multiple related copies of eukaryotic genes -- 7.6.2 Mechanism of tandem gene duplication -- 7.6.3 Pseudogenes -- 7.6.4 Concerted evolution of duplicated genes -- 7.7 Transposition of DNA -- 7.7.1 Transposable elements -- 7.7.2 Transposition in prokaryotes -- 7.7.3 Transposition in eukaryotes -- 7.8 Gene conversion -- 7.8.1 Yeast mating-type locus -- 7.8.2 Variant surface glycoprotein (VSG) genes in trypanosomes -- 7.9 Gene rearrangements -- 7.9.1 Immunoglobulin genes -- 7.9.2 T-cell receptor genes -- 7.9.3 Other gene rearrangements -- 7.10 Chromosomal translocations -- References -- 8 RNA biosynthesis -- 8.1 DNA-dependent RNA polymerases -- 8.1.1 Bacterial DNA-dependent RNA polymerase -- 8.1.2 Eukaryotic DNA-dependent RNA polymerases -- 8.2 Prokaryotic RNA synthesis -- 8.2.1 Prokaryotic initiation of transcription -- 8.2.2 Elongation of RNA transcripts -- 8.2.3 Termination of transcription in prokaryotes -- 8.3 Eukaryotic RNA synthesis -- 8.3.1 Initiation by RNA polymerase II -- 8.3.2 Initiation by RNA polymerase III -- 8.3.3 Initiation by RNA polymerase I -- 8.3.4 Eukaryotic termination -- 8.3.5 Transcription of mitochondrial and chloroplast genes -- 8.4 RNA polymerases and RNA synthesis in DNA viruses -- 8.5 The replication of RNA viruses by RNA-dependent RNA polymerase (Replicase) -- 8.5.1 RNA bacteriophage -- 8.5.2 Eukaryotic RNA viruses -- References -- 9 The arrangement of genes, their transcription and processing -- 9.1 Transcription and processing of prokaryotic and bacteriophage mRNA -- 9.2 The organization of eukaryotic protein-encoding genes -- 9.2.1 Genes are often discontinuous -- 9.2.2 Gene families and gene clustering -- 9.3 Transcription and processing of eukaryotic pre-messenger RNA -- 9.3.1 The nature of gene transcripts -- 9.3.2 Caps and 5?-leader sequences of eukaryotic mRNA -- 9.3.3 Poly adenylate tails, 3? -processing and 3? -non-coding sequences of eukaryotic mRNAs -- 9.3.4 Removal of intron transcripts from pre-mRNA -- 9.4 The arrangement of rRNA genes, their transcription and processing -- 9.4.1 The prokaryotic rRNA genes and their processing -- 9.4.2 The rRNA genes of eukaryotes -- 9.4.3 The transcription and processing of eukaryotic ribosomal RNA -- 9.5 The arrangement and expression of tRNA genes -- 9.5.1 tRNA genes -- 9.5.2 The processing of tRNA -- 9.6 The arrangement and expression of mitochondrial and chloroplast genes -- 9.6.1 Protein-encoding genes of mitochondria and chloroplasts -- 9.6.2 Mitochondrial and chloroplast rDNA -- 9.6.3 Mitochondrial and chloroplast tRNA genes -- 9.6.4 The introns of mitochondrial genes and their splicing -- 9.7 A postscript on splicing -- References -- 10 Control of transcription and mRNA processing -- 10.1 The regulation of prokaryotic RNA chain initiation -- 10.1.1 Induction of the lac operon - a negative control system -- 10.1.2 Repression of the trp operon -- 10.1.3 Catabolite repression - a positive control system -- 10.1.4 Other variations in the control of initiation at bacterial operons -- 10.1.5 The repressors of bacteriophage lambda (phage ?) -- 10.1.6 The interaction of repressor and activator proteins with DNA -- 10.2 The regulation of the termination of transcription in prokaryotes -- 10.2.1 Attenuation -- 10.2.2 Antiterminators of transcription -- 10.3 Modification of prokaryotic RNA polymerase -- 10.3.1 Diversity in sigma factor -- 10.3.2 Bacteriophage T4 modulation of host RNA polymerase -- 10.4 Control of gene expression in eukaryotes -- 10.4.1 Promotors -- 10.4.2 Cis-acting control elements -- 10.4.3 Trans-acting factors -- 10.4.4 The nature of active chromatin -- 10.4.5 Multiple gene copies, amplification and gene rearrangement -- 10.5 Regulation of gene expression by RNA -- 10.5.1 Antisense RNA -- 10.5.2 Identifiers -- 10.6 The control of pre-mRNA processing -- 10.6.1 3?-Proce.
This is the peer reviewed version of the following article: Martínez-Calvo, M.; Guerrini, L.; Rodríguez, J.; Álvarez Puebla, R. A.; Mascareñas, J. L. (2020), Surface-enhanced Raman Scattering Detection of Nucleic Acids exhibiting Sterically Accessible Guanines using Ruthenium-polypyridyl Reagents. J. Phys. Chem. Lett., 11: 7218–7223, which has been published in final form at https://doi.org/10.1021/acs.jpclett.0c02148. This article may be used for non-commercial purposes in accordance with ACS Terms and Conditions for Use of Self-Archived Versions ; Here, we report the application of surface-enhanced Raman scattering (SERS) spectroscopy as a rapid and practical tool for assessing the formation of coordinative adducts between nucleic acid guanines and ruthenium polypyridyl reagents. The technology provides a practical approach for the wash-free and quick identification of nucleic acid structures exhibiting sterically accessible guanines. This is demonstrated for the detection of a quadruplex-forming sequence present in the promoter region of the c-myc oncogene, which exhibits a nonpaired, reactive guanine at a flanking position of the G-quartets ; We are thankful for the financial support from the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2019-2022, ED431G 2019/03) and the European Union (European Regional Development Fund – ERDF). We also acknowledge the support given by the Spanish Grant SAF2013-41943-R and SAF2016-76689-R, the Xunta de Galicia (Grants 2015-CP082, ED431C 2017/19,), the Spanish Ministry de Economia y Competitividad (CTQ2017-88648R and RYC-2016-20331), the Generalitat de Cataluña (2017SGR883), the Universitat Rovira i Virgili (2019PFR-URV-B2-02), the Universitat Rovira i Virgili and Banco Santander (2017EXIT-08), and the European Research Council (Advanced Grant No. 340055). M.M.-C. thanks the Ministerio de Economı́a y Competitividad for the Postdoctoral fellowship (IJCI-2014-19326) and the Ministerio de Ciencia e Innovación and Ministerio de Universidades for ...
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The properties of water at the nanoscale are crucial in many areas of biology, but the confinement of water molecules in sub-nanometre channels in biological systems has received relatively little attention. Advances in nanotechnology make it possible to explore the role played by water molecules in living systems, potentially leading to the development of ultrasensitive biosensors. Here we show that the adsorption of water by a self-assembled monolayer of single-stranded DNA on a silicon microcantilever can be detected by measuring how the tension in the monolayer changes as a result of hydration. Our approach relies on the microcantilever bending by an amount that depends on the tension in the monolayer. In particular, we find that the tension changes dramatically when the monolayer interacts with either complementary or single mismatched single-stranded DNA targets. Our results suggest that the tension is mainly governed by hydration forces in the channels between the DNA molecules and could lead to the development of a label-free DNA biosensor that can detect single mutations. The technique provides sensitivity in the femtomolar range that is at least two orders of magnitude better than that obtained previously with label-free nanomechanical biosensors and with label-dependent microarrays. ; D.R. acknowledges the fellowship funded by the Autonomic Community of Madrid (CAM). J.T, M.C, J.M and D.R acknowledge financial support by Spanish Ministry of Science (MEC) under grant No. TEC2006-10316 and CAM under grant No. 200550M056. C.B. acknowledges funding provided by MEC under grant No. BIO2007-67523. Work at Centro de Astrobiología was supported by European Union (EU), Instituto Nacional de Técnica Aeroespacial (INTA), MEC and CAM. All the authors acknowledge A. Cebollada, J.M. García-Martín, J. García, J.L. Costa-Kramer, M. Arroyo-Hernández and J.V. Anguita for their assistance in the gold deposition on the cantilevers. ; Peer reviewed
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