Nucleic Acid Analysis Research Articles

Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment

BackgroundSurveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes.MethodsThe diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests.ResultsOf the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment.ConclusionMalaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.

Observing Mesoscopic Nucleic Acid Capacitance Effect and Mismatch Impact via Graphene Transistors.

This work reports a molecular-scale capacitance effect of the double helical nucleic acid duplex structure for the first time. By quantitatively conducting large sample measurements of the electrostatic field effect using a type of high-accuracy graphene transistor biosensor, an unusual charge-transport behavior is observed in which the end-immobilized nucleic acid duplexes can store a part of ionization electrons like molecular capacitors, other than electric conductors. To elucidate this discovery, a cascaded capacitive network model is proposed as a novel equivalent circuit of nucleic acid duplexes, expanding the point-charge approximation model, by which the partial charge-transport observation is reasonably attributed to an electron-redistribution behavior within the capacitive network. Furthermore, it is experimentally confirmed that base-pair mismatches hinder the charge transport in double helical duplexes, and lead to directly identifiable alterations in electrostatic field effects. The bioelectronic principle of mismatch impact is also self-consistently explained by the newly proposed capacitive network model. The mesoscopic nucleic acid capacitance effect may enable a new kind of label-free nucleic acid analysis tool based on electronic transistor devices. The in situ and real-time nucleic acid detections for virus biomarkers, somatic mutations, and genome editing off-target may thus be predictable.

Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material

The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.

Biofluids manipulation methods for liquid biopsy in minimally-invasive assays

The Liquid Biopsy (LB) is an opportunity for non-invasive diagnosis and prognosis of various diseases. To date, it isn't possible to consider that tissue biopsy can represent a pathology entirety. Then, body fluids are rich in a large number and variety of biomarkers and they can provide information about several diseases.•Recently, other biological fluids, easy to be collected are rising for their significant content of biomarkers and for the possibility to collect and manipulate them without the intervention of medical staff.•The management of biological fluids requires suitable storage methods. Temperature, storage time and physical stresses due to sample handling can lead to chemical and physical changes that may induce sample degradation and incorrect analysis.•The reliability of a diagnostic or screening test depends on its sensitivity and specificity. As the liquid biopsy is a 'snapshot' of a pathophysiological condition, it is crucial that its components do not degrade due to the improper handling of the body fluid.In this review, some handling methods of Saliva, Urine, Stool, Seminal Fluid, Tears and Sweat samples will be described, as well as protocols to facilitate the analysis of metabolites, nucleic acids, proteins and Extracellular Vesicles (EVs) from those unusual body fluids.

Fluorescence based miniaturized microfluidic and nanofluidic systems for biomedical applications.

Over the last two decades miniaturized microfluidic and nanofluidic systems with fluorescence setup emerged as a powerful technological platform for diverse biomedical applications. Bio-macromolecules such as nucleic acids and proteins are the core cellular components, their single molecule analysis allow us to understand biological processes, disease creation and progression, and development of novel treatment policies. Design and development of foolproof treatment methods requires rigorously analysis of nucleic acids and proteins such as length quantifications, sequence profiling, sequence mapping, analysis of conformational changes, analysis and recognition of epigenetic changes, and their interactions with other biomolecules. Miniaturized microfluidic and nanofluidic systems with fluorescence spectroscopy enable worldwide researchers to perform nucleic acids and proteins extractions and single molecule analysis from the trace amount of biological samples. In the present chapter we mostly highlighted over one decade applications of microfluidic and nanofluidic systems for single cell micro ribonucleic acid (miRNA) isolation and detection, deoxyribonucleic acid (DNA) mapping, DNA barcoding, identification of epigenetic mark on single DNA molecule, DNA-protein interactions study, protein sensing, protein sequencing, protein binding kinetics and many other applications. We also presented the recently reported microfluidic platform for the preparation of reproducible unisize aggregation induced emission (AIE) active nanomaterials and their biological applications.

Performance Comparison of GeneXpert MTB/RIF, Gene Chip Technology, and Modified Roche Culture Method in Detecting Mycobacterium tuberculosis and Drug Susceptibility in Sputum.

Our aim of this study was to observe and analyze the performance of the real-time fluorescence quantitative nucleic acid amplification detection of Mycobacterium tuberculosis/rifampicin resistance (GeneXpert MTB/RIF), gene chip technology, and modified Roche culture method in detecting MTB in sputum submitted for inspection and drug susceptibility. Patients with smear-negative suspected pulmonary TB (n = 120) in our hospital were enrolled in this study using a random number table, and sputum samples submitted for inspection were tested by the GeneXpert MTB/RIF, gene chip technology, and modified Roche culture method. With clinical diagnosis as the gold standard, the performance (mainly sensitivity and specificity) of the above three detection methods in the diagnosis of MTB was compared. Next, the drug susceptibility test (DST) was carried out on sputum samples, tested positive by the three methods. With the solid culture results as the evaluation criteria, the performance of the three detection methods in the diagnosis MTB and DST was compared. When compared with the modified Roche culture method, the GeneXpert MTB/RIF had the highest positive rate and a shorter overall test duration (P < 0.05). In contrast with the gene chip technology, the GeneXpert MTB/RIF exhibited higher sensitivity and negative predictive value (NPV) and lower specificity, accuracy, positive predictive value (PPV), and Kappa value (P < 0.05). According to analysis of the diagnostic performance of the three detection methods, GeneXpert MTB/RIF displayed the highest diagnostic sensitivity, ideal predictive values, and the highest similarity with clinical diagnosis in results (P < 0.05). The detection of susceptibility to isoniazid (INH) and RIF showed that the GeneXpert MTB/RIF and gene chip technology performed ideally in DST of MTB. In comparison with the modified Roche culture method, the GeneXpert MTB/RIF and gene chip technology have more prominent performance in detecting MTB and drug susceptibility. Besides, to further improve the accuracy of clinical diagnosis, various molecular biology detection methods can be combined to avoid delaying of the best time for the diagnosis and treatment of the disease.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

Integrated Dual Nucleic Acid Analysis by Slip Valve-Assisted Fluidic Chip Combined with CRISPR/Cas12a Detection

Integrated Dual Nucleic Acid Analysis by Slip Valve-Assisted Fluidic Chip Combined with CRISPR/Cas12a Detection

Highly accelerated isothermal nucleic acid amplifications by butanol dehydration: simple, more efficient, and ultrasensitive.

Enzyme-free isothermal amplification reactions for nucleic acid analysis usually take several hours to obtain sufficient detection sensitivity, which limits their practical applications. Herein, we report a butanol dehydration-based method to greatly improve both the efficiency and the sensitivity of nucleic acid detections by three types of enzyme-free isothermal amplification reactions. The reaction time has been shortened from 3 h to 5-20 min with higher sensitivities. Especially in the DNAzyme-based amplification, the detection limit can be lowered over 16 000-fold to 3 × 10-17 mol L-1 in 2 h compared to the normal 3 h-reaction. We demonstrate that the high amplification efficiencies are attributed to the greatly accelerated reaction rates in the extremely concentrated reaction solutions caused by the butanol dehydration. This approach enhances the potential of applications of isothermal amplification reactions in clinical rapid tests, nanostructure synthesis, etc. and is promising to expand to other types of chemical reactions.

Optical Design of a Quantitative Microvolume Nucleic Acid Spectrophotometer with Non-Optical Fiber and All Radiation-Hardened Lens Elements

The purity of the nucleic acid samples obtained by extraction/precipitation or adsorption chromatography must be verified with microvolume spectrophotometry to ensure a high success rate of the subsequent nucleic acid sequencing while exploring the trace rare nucleic acids in space exploration with in-situ life detection. This paper reports an optical design for a radiation-hardened quantitative microvolume spectrophotometer with all radiation-hardened lens elements for space exploration instruments by using a non-optical fiber optical path with radiation-hardened optical glass elements. The results showed that the mean absolute error rate of the measured standard ribonucleic acid samples at concentrations between 50 ng/μL and 2300 ng/μL was within 2% when compared with a LINKO LKU–6000 ultraviolet–visible spectrophotometer.

Multianalyte lateral flow immunoassay for simultaneous detection of protein-based inflammation biomarkers and pathogen DNA

The detection of multiple biomolecule classes in one go is highly desirable for a wide variety of areas, and in particular for point-of-care diagnostics. For example, wound infections are a major problem for patient’s health and cause huge efforts in our healthcare system. In this regard, monitoring infected wounds through simultaneous detection of pathogens via nucleic acid analysis and detection of local inflammation biomarkers is key in order to enable a personalized therapy, improve the clinical outcome and thus, leading to a reduction of overall healthcare costs. In this regard, wound exudate offers an attractive sample material which can be collected in a non-invasive manner. Here, we report the development of a Multianalyte-Assay detecting inflammation biomarkers and pathogen DNA simultaneously from one sample within 35 min. Protein-compatible amplification and labeling transforms nucleic acid information into the measurement principle for protein detection. The combination with rapid detection via lateral flow immunoassay enables a fast and straightforward analysis of multiple biomolecule classes using identical assay conditions. To demonstrate the feasibility of the Multianalyte-Assay, the proinflammatory cytokine interleukin-6 (IL-6) and gDNA of the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) are used. The detection limits of 4 ng/mL IL-6 and 70 copies/reaction P. aeruginosa gDNA meet the clinically relevant range and thus, having tremendous potential to improve the wound management at the point-of-care.

Molecular Diagnostics for Ocular Infectious Diseases: LXXVIII Edward Jackson Memorial Lecture

To review the use of molecular diagnostic techniques in the management of ocular infectious disease. Retrospective review. A combination of literature review and personal recollections are used. Although the broad term molecular diagnostics may encompass techniques to identify pathogens via protein or metabolomic signatures, this review concentrates on detection of pathogen nucleic acid as an indicator of infection. The introduction of the polymerase chain reaction (PCR) in 1985 opened a new era in analysis of nucleic acids. This technique was soon applied to the detection of potential pathogen DNA and RNA, including viruses, bacteria, and parasites in infectious eye disease. Advances in PCR have allowed class-specific diagnostics (ie, pan-bacterial and pan-fungal), quantitation of pathogen DNA, and multiplexed testing. The Human Genome Project in the early 2000s greatly accelerated development of DNA sequencers, ushering in the era of "Next Generation Sequencing" and permitting pathogen-agnostic methods for the detection of potential infectious agents. Most recently, new technologies such as nanopore sequencing have reduced both cost and equipment requirements for whole-genome sequencing; when coupled with real-time sequence analysis methods, these methods offer the promise of true, real-time, point-of-service ocular infectious disease diagnostics. Molecular methods for pathogen detection have greatly advanced the diagnosis of ocular infectious disease. Further methodologic advances will have a direct impact on the management of these conditions.

Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy.

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and 19 F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and 19 F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and 19 F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa (P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

Simultaneous Targeted Analysis of GGT and Its H-Type mRNA in HepG2 Cells Based on Degradable Silicon Nanomaterials.

Most important physiological processes in live cells are usually maintained by the interaction of multiple related biomolecules; the multi-target simultaneous analysis of these related molecules can better reflect the dynamic changes of their biological regulatory processes, providing more comprehensive information for diseases diagnosis and research. Herein, we have constructed the degradable multifunctional silica nanomaterials from the prepared degradable organic silicon source and further established degradable composite nanoprobes (DCNPs). The low toxicity of DCNPs could reduce the impact on normal physiological processes in cells and achieve the needs of living cell analysis applications; by the loading of the gamma-glutamyltransferase (GGT) activity-identification probe (Cy-GGT) and surface nucleic acid-recognizing molecular beacon (hairpin) modification, the DCNP realized the simultaneous image analysis of GGT and its related H-type mutated GGT mRNA (H-mRNA) in HepG2 cells and their quantitative detection in vitro. Compared with the traditional multi-target analysis strategy, the lack of targets' physiological mechanism connection was improved, and the combined application of small molecular probes and nucleic acid analysis structures was realized under the control of the cross-influence. This strategy is expected to provide a new direction for the design of multi-target analysis in live cells and provide more accurate analytical tools for clinical research and cancer therapy.

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, −2 and −3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.

CRISPR/Cas12a Powered DNA Framework‐Supported Electrochemical Biosensing Platform for Ultrasensitive Nucleic Acid Analysis (Small Methods 12/2021)

Front Cover In article number 2100935, Song, Ding, and co-workers have developed a CRISPR/Cas12a powered DNA framework-supported biosensing platform via cleavage of Cas12a on the heterogeneous electrochemical interface. The 3D tetrahedral framework nucleic acids would improve and expand the application of CRISPR/Cas's technology to the electrochemical interface-based nucleic acids analysis.

An In Silico Detection of a Citrus Viroid from Raw High-Throughput Sequencing Data.

E-probe Diagnostic for Nucleic Acid Analysis (EDNA) is a user-friendly bioinformatic tool that has been adapted for the detection and identification of citrus exocortis viroid (CEVd). Here, we describe the procedures for RNA extraction from citrus tissues, library and sequencing preparation, and the utilization of EDNA Mi-Finder online platform on raw high-throughput sequencing (HTS) data.

Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip.

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100molecules/μL showed a strong correlation (R2=0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.