Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

A dual paper-based nucleic acid extraction method from blood in under ten minutes for point-of-care diagnostics.

Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.

Rapid automatic nucleic acid purification system based on gas-liquid immiscible phase.

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas-liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%-95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.

Improving the specificity of nucleic acid detection with endonuclease-actuated degradation.

Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat.

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.

Magnetic-Responsive Surface-Enhanced Raman Scattering Platform with Tunable Hot Spot for Ultrasensitive Virus Nucleic Acid Detection.

Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.

Solid-phase extraction methods for nucleic acid separation. A review.

The separation and purification of biomacromolecules such as nucleic acid is a perpetual topic in separation processes and bioengineering (fine chemicals, biopharmaceutical engineering, diagnostics, and biological characterization). In principle, the solid-phase extraction for nucleic acid exhibits efficient phase separation, low pollution risk, and small sample demand, compared to the conventional liquid-phase extraction. Herein, solid-phase extraction methods are systematically reviewed to outline research progress and explore additional solid-phase sorbents and devices for novel, flexible, and high-efficiency nucleic acid separation processes. The functional materials capture nucleic acid, magnetic and magnetic-free solid-phase extraction methods, separation device design and optimization, and high-throughput automatable applications based on high-performance solid-phase extraction are summarized. Finally, the current challenges and promising topics are discussed.

Performance Accuracy of a Laboratory-Developed Real-Time RT-PCR Method for Detection of SARS-CoV-2 in Self-Collected Saliva Specimens.

OBJECTIVE: The ongoing COVID-19 pandemic caused by SARS-CoV-2 has challenged diagnostic laboratories to re-examine traditional methods for collecting specimens and sample types used in molecular testing. Our goal was to demonstrate that saliva can be used for detecting SARS-CoV-2 and correlates well with established molecular methods using nasopharyngeal (NP) swabs. METHODS: We examined use of a saliva collection device in conjunction with a laboratory-developed real-time reverse transcription-polymerase chain reaction (LDPCR) method for detecting SARS-CoV-2 in a symptomatic population and compared results with 2 US Food and Drug Administration (FDA)-approved methods (emergency use authorization [EUA]) that use specimens from NP swabs. RESULTS: The sensitivity of LDPCR compared with the reference methods was 75.0% (21/28); specificity, 98.1% (104/106). When cycle threshold values were compared between paired specimens using the LDPCR and a EUA reverse transcription PCR method, both targeting the open-reading frame gene, the mean value for saliva was 4.66 cycles higher than for NP specimens. CONCLUSION: Use of self-collected saliva in conjunction with an LDPCR for SARS-CoV-2 compared favorably with 2 FDA EUA methods using NP swabs. The use of an alternative sample type and assay method will aid in expanding the availability of testing during the ongoing COVID-19 pandemic.

Nucleic acid extraction without electrical equipment via magnetic nanoparticles in Pasteur pipettes for pathogen detection.

The outbreak of COVID-19 makes epidemic prevention and control become a growing global concern. Nucleic acid amplification testing (NAAT) can realize early and rapid detection of targets, thus it is considered as an ideal approach for detecting pathogens of severe acute infectious diseases. Rapid acquisition of high-quality target nucleic acid is the prerequisite to ensure the efficiency and accuracy of NAAT. Herein, we proposed a simple system in which magnetic nanoparticles (MNPs) based nucleic acid extraction was carried out in a plastic Pasteur pipette. Different from traditional approaches, this proposed system could be finished in 15 min without the supports of any electrical instruments. Furthermore, this system was superior to traditional MNPs based extraction methods in the aspects of rapid extraction and enhancing the sensitivity of a NAAT method, accelerated denaturation bubbles mediated strand exchange amplification (ASEA), to the pathogens from various artificial samples. Finally, this Pasteur pipette system was utilized for pathogen detection in actual samples of throat swabs, cervical swabs and gastric mucosa, the diagnosis results of which were identical with that provided by hospital. This rapid, easy-performing and efficiency extraction method ensures the applications of the NAAT in pathogen detection in regions with restricted resources.

Establishing an internal quality control method for the stable extraction of nucleic acids of severe acute respiratory syndrome coronavirus 2 and RT-PCR-based detection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is detected using real-time RT-PCR. However, there are limitations pertaining to quality control, particularly with respect to establishing quality control measures for extraction of viral nucleic acids. Here, we investigated the quality control measures for the various processes using an extrinsic quality control substance and quality control charts. METHODS: An extrinsic quality control substance was added to the sample, and then, real-time RT-PCR was performed. Samples with negative test results and the corresponding data were analyzed; a quality control chart was created and examined. RESULTS: Data analysis and the quality control charts indicated that SARS-CoV-2 could be reliably detected using real-time RT-PCR, even when different nucleic acid extraction methods were used or when different technicians were employed. CONCLUSION: With the use of quality control substances, it is possible to achieve quality control throughout the process-from nucleic acid extraction to nucleic acid detection-even upon using varying extraction methods. Further, generating quality control charts would guarantee the stable detection of SARS-CoV-2.

Rapid and simple single-chamber nucleic acid detection system prepared through nature-inspired surface engineering.

Background: Nucleic acid (NA)-based diagnostics enable a rapid response to various diseases, but current techniques often require multiple labor-intensive steps, which is a major obstacle to successful translation to a clinical setting. Methods: We report on a surface-engineered single-chamber device for NA extraction and in situ amplification without sample transfer. Our system has two reaction sites: a NA extraction chamber whose surface is patterned with micropillars and a reaction chamber filled with reagents for in situ polymerase-based NA amplification. These two sites are integrated in a single microfluidic device; we applied plastic injection molding for cost-effective, mass-production of the designed device. The micropillars were chemically activated via a nature-inspired silica coating to possess a specific affinity to NA. Results: As a proof-of-concept, a colorimetric pH indicator was coupled to the on-chip analysis of NA for the rapid and convenient detection of pathogens. The NA enrichment efficiency was dependent on the lysate incubation time, as diffusion controls the NA contact with the engineered surface. We could detect down to 1×103 CFU by the naked eye within one hour of the total assay time. Conclusion: We anticipate that the surface engineering technique for NA enrichment could be easily integrated as a part of various types of microfluidic chips for rapid and convenient nucleic acid-based diagnostics.

Cellulose supported magnetic nanohybrids: Synthesis, physicomagnetic properties and biomedical applications-A review.

Cellulose and its forms are widely used in biomedical applications due to their biocompatibility, biodegradability and lack of cytotoxicity. It provides ample opportunities for the functionalization of supported magnetic nanohybrids (CSMNs). Because of the abundance of surface hydroxyl groups, they are surface tunable in either homogeneous or heterogeneous solvents and thus act as a substrate or template for the CSMNs' development. The present review emphasizes on the synthesis of various CSMNs, their physicomagnetic properties, and potential applications such as stimuli-responsive drug delivery systems, MRI, enzyme encapsulation, nucleic acid extraction, wound healing and tissue engineering. The impact of CSMNs on cytotoxicity, magnetic hyperthermia, and folate-conjugates is highlighted in particular, based on their structures, cell viability, and stability. Finally, the review also discussed the challenges and prospects of CSMNs' development. This review is expected to provide CSMNs' development roadmap in the context of 21st-century demands for biomedical therapeutics.

Sticky problems: extraction of nucleic acids from molluscs.

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the 'hidden break' in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis.

Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Macroporous Polymer Monoliths for Affinity Chromatography and Solid-Phase Enzyme Processing.

Nowadays, monolithic stationary phases, because of their special morphology and enormous permeability, are widely used for the development and realization of fast dynamic and static processes based on the mass transition between liquid and solid phases. These are liquid chromatography, solid-phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including the bioaffinity mode, represents unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In the edited chapter, the examples of the application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by the combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents the original and practically valuable method that can be used in biotechnology. In addition, macroporous monoliths were adapted to the immobilization of such different enzymes as polynucleotide phosphorylase, ribonuclease A, α-chymotrypsin, chitinolytic biocatalysts, ß-xylosidase, and ß-xylanase. The possibility of use of immobilized enzyme reactors based on monoliths for different purposes is demonstrated.

Tissue Preservation and FFPE Samples: Optimized Nucleic Acids Isolation in Ewing Sarcoma.

Different methods have been described for the preservation of biopsy or resection samples. In the routine pathology, the cheapest and most commonly used is fixation of samples in formalin and embedding in paraffin (FFPE samples). This method preserves tissue samples for a very long time and is suitable for several specialized techniques such as fluorescence in situ hybridization (FISH) and immunohistochemistry, the latter being the most frequent and often the only additional method used for establishment of final diagnosis. However, in light of the growing need of next-generation sequencing and microarray technologies that are often very helpful to establish and/or confirm diagnoses in the field of pediatric sarcoma (including Ewing sarcoma), preservation of high-quality and quantity of nucleic acids (DNA/RNA) is desirable. Herein, we describe how to ideally preserve samples, as well as how to proceed to isolate nucleic acids for successful subsequent molecular assays with a special focus on Ewing sarcoma samples.

Droplet Microarray Based on Nanosensing Probe Patterns for Simultaneous Detection of Multiple HIV Retroviral Nucleic Acids.

Multiplexed detection of viral nucleic acids is important for rapid screening of viral infection. In this study, we present a molybdenum disulfide (MoS2) nanosheet-modified dendrimer droplet microarray (DMA) for rapid and sensitive detection of retroviral nucleic acids of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) simultaneously. The DMA platform was fabricated by omniphobic-omniphilic patterning on a surface-grafted dendrimer substrate. Functionalized MoS2 nanosheets modified with fluorescent dye-labeled oligomer probes were prepatterned on positively charged amino-modified omniphilic spots to form a fluorescence resonance energy transfer (FRET) sensing microarray. With the formation of separated microdroplets of sample on the hydrophobic-hydrophilic micropattern, prepatterned oligomer probes specifically hybridized with the target HIV genes and detached from the MoS2 nanosheet surface due to weakening of the adsorption force, leading to fluorescence signal recovery. As a proof of concept, we used this microarray with a small sample size (<150 nL) for simultaneous detection of HIV-1 and HIV-2 nucleic acids with a limit of detection (LOD) of 50 pM. The multiplex detection capability was further demonstrated for simultaneous detection of five viral genes (HIV-1, HIV-2, ORFlab, and N genes of SARS-COV-2 and M gene of Influenza A). This work demonstrated the potential of this novel MoS2-DMA FRET sensing platform for high-throughput multiplexed viral nucleic acid screening.

A systematic review and meta-analysis of different diagnostic procedures to detect gonococcus infection in resource-limited scenario.

Context: Neisseria gonorrhoeae is a Gram-negative diplococcus, an obligate human pathogen, and the etiologic agent of the sexually transmitted infection (STI), gonorrhoea. culture is the standard procedure for diagnosis, which may be supported by nucleic acid tests and microscopy. Aims: To determine the best possible method of diagnosis for Gonococcus infection in resource-limited settings. Settings and Design: The meta-analyses were designed to determine the difference in diagnosis between Culture and nucleic acid amplification tests (NAATs) and also between the different Amplification Tests and widely available Roche COBAS AMPLICOR test. Subjects and Methods: Databases searched were Pubmed, Medline, Google Scholar and Cochrane reviews. Risk ratio (RR) with 95% confidence intervals was estimated for the dichotomous outcomes. The random-effect model was applied for all the studies in the analysis. Statistical Analysis Used: The meta-analysis was computed in RevMan Version 5.3, Copenhagen, Denmark. Results: In the first analysis, NAATs significantly improved the chances of detection in comparison to the standard culture and final RR was 1.24 (1.05-2.51), which put the diamond on the right of no-effect axis, indicating more positives by NAATs. In the second analysis, AMPLICOR had the more positive results, which may have indicated better detection rate, as well as less specificity and final RR was 0.809 (0.737-0.888), which put the diamond on the left of the non-effect axis, indicating more positives by AMPLICOR. Conclusions: In a resource-limited scenario like India, the syndromic management of STIs are considered to be the norm. A positive diagnosis is only given if the tests are confirmed by Culture, as it is still considered to be the gold standard of diagnosis. However, in many cases, due to suboptimal transportation and lack of proper handling, culture in unable to grow even if the patient is infected. In such cases, Nucleic Acid Tests should be able to detect an infection.

Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish.

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.