Nucleic Acid Detection Research Articles

Study on the correlation between abdominal infection and psychological stress in children based on nucleic acid detection

BACKGROUND Diagnosing and treating abdominal infection in children remains a challenge. Nucleic acid detection, as a rapid and accurate diagnosis tool, has great significance in this field. AIM To investigate the diagnosis and treatment of abdominal infection by nucleic acid detection and its possible correlation with psychological stress in children. METHODS A total of 50 pediatric patients diagnosed with abdominal infections between September 2020 and July 2021 were included in this study. Intra-abdominal pus samples were collected for pathogen culture, drug susceptibility testing, and broad-spectrum bacterial nucleic acid testing. Psychological stress, anxiety, depression, and coping styles were assessed using the coping with a disease (CODI) scale. RESULTS Based on susceptibility testing, a regimen of cefazoxime, piperacillin/tazobactam, and metronidazole or ornidazole achieved 100% effectiveness in treating appendicitis. Psychological assessments revealed a positive correlation between pressure level and both anxiety (r = 0.324, P = 0.001) and depressive disorders (r = 0.325, P < 0.001). Acceptance and distancing as coping strategies were negatively correlated with anxiety and depression, while negative emotional responses were strongly associated with increased anxiety (r = 0.574, P < 0.001) and depression (r = 0.511, P = 0.001). Coping strategies such as illusion and escape showed no significant correlation with emotional outcomes. CONCLUSION Nucleic acid testing helps in the diagnosis of abdominal infections in children, and also focuses on children's mental health.

Musculoskeletal Disorders in COVID-19 Surveillance Staff Members: An Analysis of Prevalence, Risk Factors, and Coping Strategies

Aims/Background During the coronavirus disease 2019 (COVID-19) pandemic, the COVID-19 surveillance staff members experienced an increased risk for musculoskeletal disorders. Thus, it is necessary to further investigate the causal factors stemming from COVID-19 surveillance work, especially nucleic acid testing, and establish their relationship with work-related musculoskeletal disorders (WMSDs) by building upon the previous research. This study aimed to determine the prevalence of WMSDs and the major risks faced by the COVID-19 surveillance staff members. Methods This cross-sectional study was conducted in May 2022. The Quick Exposure Check (QEC), Nordic Musculoskeletal Questionnaire (NMQ), and Maslach Burnout Inventory (MBI) were used to evaluate ergonomic factors, musculoskeletal symptoms and pressure among 131 COVID-19 surveillance staff members in four hospitals in South-central Henan Province. Additionally, data regarding the coping strategies adopted by the respondents for lowing the risk of developing WMSDs were collected using a self-report questionnaire. Results The QEC results showed that the back, neck, and should/arm of the COVID-19 surveillance staff members bore high ergonomic loads, while the wrist suffered a medium load. The neck (75.6%), shoulder (66.4%), upper back (58%), and wrist (55.7%) were at higher risk for musculoskeletal disorder. Most of the respondents claimed a low sense of achievement (124, 94.7%) and slightly less than half of them had endured emotional exhaustion at work (61, 46.6%). Prolonged period of maintaining the same posture at work, awkward bending or twisting of back side, task repetition and so on were the perceived job risk factors precipitating WMSDs among the participants in this study. The top three coping strategies for lowering the risk of WMSDs included adjusting the plinth/bed height (49.6%), utilizing different parts of the body at work (47.3%), and taking short breaks when required (33.6%). Conclusion Prolonged large-scale nucleic acid detection can aggravate the musculoskeletal injuries of the neck, shoulder, upper back, and wrist in COVID-19 surveillance staff members. Prevention measures for musculoskeletal injuries factors must be in place to deter such incidence at work.

Split Probe-Induced Protein Translational Amplification for Nucleic Acid Detection.

Nucleic acid detection is important in a wide range of applications, including disease diagnosis, genetic testing, biotechnological research, environmental monitoring, and forensic science. However, the application of nucleic acid detection in various fields is hindered by the lack of sensitive, accurate, and inexpensive methods. This study introduces a simple approach to enhance the sensitivity for the accurate detection of nucleic acids. Our approach combined a split-probe strategy with in vitro translational amplification of reporter protein for signal generation to detect nucleic acids with high sensitivity and selectivity. This approach enables target-mediated translational amplification of reporter proteins by linking split probes in the presence of a target microRNA (miRNA). In particular, the fluorescence split-probe sensor adopts a reporter protein with various fluorescence wavelength regions, enabling the simultaneous detection of multiple target miRNAs. Moreover, luminescence detection by merely altering the reporter protein sequence can substantially enhance the sensitivity of detection of target miRNAs. Using this system, we analyzed and quantified target miRNAs in the total RNA extracted from cell lines and cell-derived extracellular vesicles with high specificity and accuracy. This split-probe sensor has potential as a powerful tool for the simple, sensitive, and specific detection of various target nucleic acids.

Efficient and highly amplified imaging of nucleic acid targets in cellular and histopathological samples with pSABER.

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate covisualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed 'pSABER', for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide, enabling the localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets in cultured cells and FFPE specimens. Furthermore, pSABER can achieve fivefold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

A Novel High-Throughput Sample-in-Result-Out Device for the Rapid Detection of Viral Nucleic Acids

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

Development of Triggerable Peroxidase-Tagged Primers for Colorimetric DNA Detection Applicable to Various DNA Polymerases

Abstract To meet the demand for more cost-effective rapid and sensitive nucleic acid detection methods to benefit various applications, we introduce a novel technique that uses a modified triggerable guanine quadruplex-based peroxidase (POD)-tagged primer. This technique allows sensitive colorimetric detection by activating an inactive POD tag during PCR amplification, which, importantly, functions independently of the type of DNA polymerase used.

End-point diagnostics of Giardia duodenalis assemblages A and B by combining RPA with CRISPR/Cas12a from human fecal samples.

Giardia duodenalis is a common enteric protozoan parasite that is categorized into eight assemblages (A-H). In particular, assemblages A and B are zoonotic, capable of infecting both humans and animals worldwide, resulting in significant economic losses and public health challenges in epidemic regions. Thus, the development of rapid, accurate and non-laboratory-based diagnostic methods for infected animals is crucial for the effective prevention and control of giardiasis. Recent advancements in clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein (Cas12a) systems allow promising avenues for nucleic acid detection, characterized by their high flexibility, sensitivity and specificity. Combined recombinase polymerase amplification and CRISPR/Cas12a systems were combined and used as end-point diagnostic methods (termed REPORT) to detect G. duodenalis assemblage A and B. The diagnostic results can be observed by fluorescence readouts with the naked eye under blue light or colorimetric signals using a lateral flow strip (LFS). The limit of detection (LOD) of the REPORT‑based G. duodenalis assemblage A detection was 2.04 CFU/ml and 10 trophozoites per gram (TPG), and the LOD of assemblage B was 1.1 CFU/ml and 10 cysts per gram (CPG). The REPORT‑based G. duodenalis assemblage A and assemblage B detection methods have strong specificity and no cross-reactivity with other assemblages of G. duodenalis or common enteric parasitic protozoa and have excellent performance in clinical sample detection. This study presents a novel strategy for the direct identification of G. duodenalis assemblages A and B, requiring neither highly trained personnel nor costly specialized equipment.

Argonaute-Based Nucleic Acid Detection Technology: Advantages, Current Status, Challenges, and Perspectives.

Rapid and accurate detection is a prerequisite for precise clinical diagnostics, ensuring food safety, and facilitating biotechnological applications. The Argonaute system, as a cutting-edge technique, has been successfully repurposed in biosensing beyond the CRISPR/Cas system (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins), which has been extensively researched, but recognition of PAM sequences remains restricted. Argonaute, as a programmable and target-activated nuclease, is repurposed for fabricating novel detection methods due to its unparalleled biological features. In this comprehensive review, we initially elaborate on the current methods for nucleic acid testing and programmable nucleases, followed by delving into the structure and nuclease activity of the Argonaute system. The advantages of Argonaute compared with the CRISPR/Cas system in nucleic acid detection are highlighted and discussed. Furthermore, we summarize the applications of Argonaute-based nucleic acid detection and provide an in-depth analysis of future perspectives and challenges. Recent research has demonstrated that Argonaute-based biosensing is an innovative and rapidly advancing technology that can overcome the limitations of existing methods and potentially replace them. In summary, the implementation of Argonaute and its integration with other technologies hold promise in developing customized and intelligent detection methods for nucleic acid testing across various aspects.

Binding-driven forward tearing protospacer activated CRISPR-Cas12a system and applications for microRNA detection

CRISPR-Cas12a system, characterized by its precise sequence recognition and cleavage activity, has emerged as a powerful and programmable tool for molecular diagnostics. However, current CRISPR-Cas12a-based nucleic acid detection methods, particularly microRNA (miRNA) detection, necessitate additional bio-engineering strategies to exert control over Cas12a activity. Herein, we propose an engineered target-responsive hairpin DNA activator (TRHDA) to mediate forward tearing protospacer activated CRISPR-Cas12a system, which enables direct miRNA detection with high specificity and sensitivity. Target miRNA specifically binding to hairpin DNA can drive forward tearing protospacer in the stem sequence of hairpin structure, facilitating the complementarity between crRNA spacer and protospacer to activate Cas12a. Upon the hairpin DNA as input-responsive activator of Cas12a, a universal biosensing method enables the multiple miRNAs (miR-21, let-7a, miR-30a) detection and also has exceptional capability in identifying single-base mismatches and distinguishing homologous let-7/miR-30 family members. Besides, TRHDA-mediated Cas12a-powered biosensing has realized the evaluation of miR-21 expression levels in diverse cellular contexts by intracellular imaging. Considering the easy programmability of hairpin DNA in responsive region, this strategy could expand for the other target molecules detection (e.g., proteins, micromolecules, peptides, exosomes), which offers significant implications for biomarkers diagnostics utilizing the CRISPR-Cas12a system toolbox.Graphical abstract

Magnetoelastic Immunosensor for the Rapid Detection of SARS-CoV-2 in Bioaerosols.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a protein-coated, single-stranded RNA virus that parasitizes and infects primates, including humans. The current detection methods are mainly carried out using humans as a medium, such as a chest computed tomography (CT) examination, nucleic acid detection, antibody detection, and antigen detection. In addition, SARS-CoV-2 in bioaerosols is an important way of transmission and infection, which has attracted wide attention. In this paper, we simulate a sampling system of SARS-CoV-2 in bioaerosols and detect SARS-CoV-2 in bioaerosols by means of a magnetoelastic (ME) sensing device based on an Android intelligent terminal, which can directly detect SARS-CoV-2 in bioaerosols in a certain unit environment. This approach aims to achieve early detection and prevention. The experimental results show that the sampling system can successfully collect SARS-CoV-2 in bioaerosols. In the linear range of 5-20 ng/mL, the ME sensing detection system results closely match those of the calibration instrument (VNA), with goodness-of-fit values of 0.997 and 0.995, respectively. This work demonstrates that the ME sensing detection system proposed is stable, highly specific, real-time, rapid, and has certain reference values and feasibility.

At the mercy of the AI algorithm - discrepant PCR results.

As pathogen molecular diagnostics technology develops, the software analysis of polymerase chain reaction (PCR) sigmoidal curves, crossing threshold (Ct) values and the limit of nucleic acid detection for that assay, is not always available when reporting test results. These are critical components in interpreting results and can affect the management of patients. Our view is that all commercial assay providers must ensure users, as they can with laboratory developed tests, are able to see the curve and Ct value for all samples. Some examples demonstrating this principle are included. Regulatory bodies have a key role to play.

Enzyme-Responsive DNA Condensates.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organize different substrates in concentric subcompartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides expanding our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

A hand-held in-situ nucleic acid detection device based on dual-function GHz acoustofluidic resonator

Microfluidic-based nucleic acid testing (NAT) has been proven as an alternative approach for in-situ and rapid NA detection. However, these on-chip methods still require complex peripherals, which contradict the purpose of point-of-care (POC) applications. One of the major challenges is the integration of NA purification and amplification in a miniaturized setup with high mass and heat transfer efficiency. Here, we propose a hand-held in-situ nucleic acid detection device featuring a dual-functional GHz acoustofluidic resonator. The device is designed to utilize GHz acoustic streaming and acoustothermal effects to address both liquid mixing and heating issues, ensuring uniform reaction conditions and consistent thermal management in the microfluidic channel. By replacing traditional magnetic beads with polystyrene beads as matrix for solid-phase microextraction, the redundant washing and elution steps are eliminated, thereby simplifying the NA extraction process. The mixing and heating functions can be seamlessly switched by adjusting the applied power, thereby enhancing NA processing without the need for additional peripherals. The developed hand-held NAT apparatus achieved a detection limit of 10³ copies per milliliter in complex plasma samples within 35 min. Our method simplifies workflows, improves NA retention, and is suitable for processing complex biological samples, making it ideal for POC and in-field testing applications.

Fully Integrated Microfluidic Digital Chip for Simple and Highly Quantitative Detection of Norovirus.

The prevalence of foodborne illnesses is a significant global concern, resulting in numerous illnesses, deaths, and substantial economic losses annually. Traditional detection methods for foodborne pathogens are often slow, limited, and impractical for field use, underscoring the need for rapid, sensitive, and portable assays. Microfluidic technology has emerged as a promising solution for sample preparation, reaction, and detection on a small scale. Our study introduces a novel microfluidic digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultrasensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward the on-site detection of foodborne pathogens, thereby enhancing food safety and mitigating disease outbreaks.

Electric-field assisted silicon nanowire field effect transistor for the ultra-low concentration nucleic acid detection

Ultra-low concentration nucleic acid detection is crucial for disease diagnosis and prognosis. Silicon nanowire field-effect transistors (SiNW FETs) are promising due to their sensitivity, real-time capabilities, and compact design. A critical consideration for FETs is the reaction time required for nucleic acid diffusion to the detection surface, especially at low concentrations. This study utilizes polycrystalline silicon nanowire FETs (poly-SiNW FETs) as biosensors, employing a negative voltage on the liquid gate used for detection to create an electric field. This field accelerates nucleic acid diffusion towards the sensor surface to interact with immobilized probes. We adjusted the gate voltages and target solution injection flow rates to identify optimal parameters for nucleic acid detection and how the electrical field accelerates hybridization kinetics. We varied the probe immobilization times to show that higher ligand density accelerates the interaction with the immobilized probe. The study demonstrated stable diffusion of target DNA by combining FET with an electric field of −1 V and a slow injection flow rate, reducing the equilibrium time from 60 to 20 min. Additional improvement was achieved by enhancing probe immobilization density and applying an electric field, resulting in a faster probe-target hybridization reaction rate. These efforts significantly improved the signal to maintain superior performance and reduced the time required to reach equilibrium. This research pioneers using an external electric field to expedite detection time in field-effect transistors, demonstrating the potential for accelerated nucleic acid detection in nanowire FETs.

A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.

The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

One Health Farming: Noninvasive monitoring reveals links between farm vertebrate richness and pathogen markers in outdoor hoofstock

Outdoor farming contributes to biodiversity conservation and enhances animal welfare, but also raises biosafety concerns due to livestock contact with potentially infected wildlife. Thus, there is a need to assess the balance between vertebrate species richness on farms, visits by wildlife species posing a biosafety risk, and pathogen circulation in open-air farming systems. We explored these links in a pilot study involving 15 open-air hoofstock farms (6 cattle, 5 small ruminant, and 4 pig farms), where we conducted interviews and risk point inspections and used two noninvasive tools: short-term camera trap (CT) deployment and environmental nucleic acid detection (ENAD). CTs were deployed to assess the richness of birds and mammals, as well as to determine the percentage of CTs detecting defined risk species. We also collected livestock feces and used sponges to sample surfaces for environmental DNA (eDNA), testing for nine pathogen markers. Total vertebrate richness ranged from 18 to 42 species, with waterholes significantly contributing to farm vertebrate richness, since 48.2 % of all wild vertebrates were detected at waterbodies, and 28.6 % were exclusively detected at waterholes. Pathogen markers detected at risk points correlated with those detected in livestock samples. Notably, the frequency of uidA marker detection correlated with the total number of pathogen markers detected per farm. Overall marker richness, an indicator of pathogen diversity, varied between farms, being higher in small ruminant farms compared to cattle or pig farms. At the farm level, wild vertebrate richness was negatively correlated with the richness of pathogen markers detected at risk points. Additionally, risk points with a higher probability of detecting more pathogen markers had lower vertebrate richness. Although CT-based assessments of vertebrate richness and ENAD-based pathogen marker detection are only indicators of actual biodiversity and farm health, respectively, our findings suggest that farmland vertebrate communities provide important ecosystem services and may help limit the circulation of multi-host pathogens.

Digital CRISPR-Powered Biosensor Concept without Target Amplification Using Single-Impact Electrochemistry.

The rapid and reliable detection and quantification of nucleic acids is crucial for various applications, including infectious disease and cancer diagnostics. While conventional methods, such as the quantitative polymerase chain reaction are widely used, they are limited to the laboratory environment due to their complexity and the requirement for sophisticated equipment. In this study, we present a novel amplification-free digital sensing strategy by combining the collateral cleavage activity of the Cas12a enzyme with single-impact electrochemistry. In doing so, we modified silver nanoparticles using a straightforward temperature-assisted cofunctionalization process to subsequently detect the collision events of particles released by the activated Cas12a as distinct current spikes on a microelectrode array. The functionalization resulted in stable DNA-AgNP conjugates, making them suitable for numerous biosensor applications. Thus, our study demonstrates the potential of clustered regularly interspaced short palindromic repeats-based diagnostics combined with impact-based digital sensing for a rapid and amplification-free quantification of nucleic acids.

A strategy for detecting CSFV using DNAzyme-HCR cascade amplification.

The Hybridization Chain Reaction (HCR) is an isothermal amplification technique widely used for sensing nucleic acids and small molecules. Despite its effectiveness, conventional linear HCR exhibits relatively slow kinetics and insufficient sensitivity. To address this challenge, we have innovatively combined HCR with DNAzyme technology to enhance nucleic acid detection. In this novel approach, the presence of a target molecule triggers the formation of DNAzyme, leading to the cleavage of substrate S, the initiation of HCR, and the production of DNA nanowires and labeled DNAzyme. The newly generated DNAzyme continuously cleaves substrate S, promoting sequential HCR amplification and significantly enhancing the fluorescence signal. This system offers a simple, sensitive, selective, and versatile method for nucleic acid detection, with a detection limit as low as 5 pM. When tested on classical swine fever virus (CSFV) samples, the system demonstrated detection accuracy comparable to RT-qPCR and exhibited superior repeatability.

CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.

Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.