Nucleic Acid Assays Research Articles

Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R2 = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

Detection of Hbv Dna By Direct Pcr Protocol And Nested Pcr Protocol And Comparing Them. In Chronic Patients and Healthy Carriers of Hepatitis B Virus.

Hepatitis B virus is a major public health problem, and the outcome of infection depends on the effectiveness of the virus's interaction with the host, and in particular on the strength of the natural and conditioned response of humoral and cellular immunity. This study was conducted to assess the clinical status, genetic immunity and viral (viral) traits of Iraqi patients with chronic hepatitis B virus and healthy carriers of hepatitis B surface antigen, as well as to show the relationship between the immune status and the chronic condition of the disease. The study included (60) patients with chronic hepatitis B virus type (B) and (60) healthy hepatitis B surface antigen carriers who attended the Teaching Hospital for Gastrointestinal and Liver Diseases in Nasiriya City Al-Hussein Teaching Hospital, Thalassemia Center and the Central Blood Bank in Nasiriya Citysouthern of Iraq. for the period from the first period. From February 2022 until the end of February 2022, the average age of patients infected with chronic hepatitis B virus was 64.5 years, and the average age of healthy carriers of hepatitis B surface antigen was 35. 1 year, and the ratio of males to females was (12.2:) for chronic patients and (13.6:) for carriers of the disease. As for the deoxyribonucleic acid, it was (100%) in chronic patients and healthy carriers using the Nested type polymerase chain reaction, and at a rate of (30.0%) and (46.7%) in chronic patients and healthy carriers, respectively using the polymerase chain reaction technique. direct with a significant difference between the two interactions. The current results confirm that a nested PCR protocol to detect HBV DNA is a more sensitive way of detecting HBV in Iraq CHB patients and carriers. Recent advanced techniques such as nucleic acid assays, quantitative assays for viral load and RT-PCR for Iraqi viral hepatitis laboratories are recommended.

Establishment of a simple, sensitive, and specific ASFV detection method based on Pyrococcus furiosus argonaute

African swine fever (ASF), which is casued by African swine fever virus (ASFV), is a fatal infectious disease of pigs that results in significant losses to the breeding industry. Therefore, screening and detection are crucial for the control and prevention of the ASFV. Argonaute is a new detection tool that is being extensively used due to its high specificity and programmability. This study reports on a new nucleic acid assay method, termed REPD, which uses recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection. One-pot REPD was developed for the detection of ASFV. The one-pot REPD could detect a single copy of ASFV nucleic acid and showed no cross-reactivity with other pathogens. Detection in clinical samples was 100% consistent with the results of real-time PCR analysis. The results showed that the one-pot REPD assay is convenient, sensitive, specific, and potentially adaptable to the detection of ASFV. In summary, this study highlights a novel method that can be employed for the detection of pathogens.

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.

Rapid and accurate SERS assay of disease-related nucleic acids based on isothermal cascade signal amplifications of CRISPR/Cas13a system and catalytic hairpin assembly

Developing rapid, accurate and convenient nucleic acid diagnostic techniques is essential for the prevention and control of contagious diseases that are prone to gene mutations and may have homologous sequences, especially emerging infectious diseases such as the SARS-CoV-2 pandemic. Herein, a one-pot SERS assay integrating isothermal cascade signal amplification strategy (i.e., CRISPR/Cas13a system (Cas13a) and catalytic hairpin assembly (CHA), Cas13a-CHA) and SERS-active silver nanorods (AgNRs) sensing chips was proposed for rapid and accurate detection of disease-related nucleic acids. Taking SARS-CoV-2 RNA assay as a model, the Cas13a-CHA based SERS sensing strategy can achieve ultra-high sensitivity low to 5.18 × 102 copies·mL−1 within 60 min, and excellent specificity, i.e., not only the ability to identify SARS-CoV-2 RNA from gene mutations, but also incompatibility with coronaviruses such as severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and other respiratory viruses. The proposed Cas13a-CHA based SERS assay for SARS-CoV-2 RNA has satisfactory sensitivity, specificity, uniformity, and repeatability, and can be easily expanded and universalized for screening different viruses, which is expected to promise as a crucial role for diagnosis of disease-related nucleic acids in various medical application scenarios.

A high-throughput DNA analysis method based on isothermal amplification on a suspension microarray for detecting mpox virus and viruses with comparable symptoms

BackgroundMpox is a zoonotic disease caused by mpox virus (MPXV) infection. Since May 2022, there has been a marked increase in human mpox cases in different regions. Rash, fever, and sore throat are typical signs of mpox. However, other viruses, such as the B virus (BV), herpes simplex virus types 1 (HSV-1), herpes simplex virus types 2 (HSV-2), and varicella zoster virus (VZV), can also infect people and cause comparable symptoms. Therefore, clinical symptoms and signs alone make distinguishing MPXV from these viruses difficult. ResultsIn this study, we combined suspension microarray technology with recombinase-aided amplification technology (RAA) to establish a high-throughput, sensitive, and quantitative method for detecting MPXV and other viruses that can cause similar symptoms. The experimental results confirmed that the technique has outstanding sensitivity, with a minimum detection limit (LOD) of 0.1 fM and a linear range of 0.3 fM to 20 pM, spanning five orders of magnitude. The approach also exhibits exquisite selectivity, as the amplified signal can only be detected when the target virus nucleic acid is present. Additionally, serum recoveries ranging from 80.52% to 119.09% suggest that the detection outcomes are generally considered reliable. Moreover, the time required for detection using this high-throughput method is very short. After DNA extraction, the detection signal amplified by isothermal amplification on the bead array can be obtained in just 1 h. Significance and noveltyOur research introduces a new technique that utilizes suspension microarray technology and isothermal amplification to create a high-throughput nucleic acid assay. This innovative method offers multiple benefits compared to current techniques, such as being cost-effective, time-efficient, highly sensitive, and having high throughput capabilities. Furthermore, the assay is applicable not only for detecting MPXV and viruses with similar symptoms, but also for clinical diagnostics, food safety, and environmental monitoring, rendering it an effective tool for screening harmful microorganisms.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate.

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Signal amplified colorimetric nucleic acid detection based on autocatalytic hairpin assembly.

Herein, a nucleic acid assay based on autocatalytic hairpin assembly (ACHA) was proposed. In this system, two split G-quadruplex sequences were integrated into H1 and H2, respectively. And a DNA strand with the same sequence to target DNA was integrated into the assistant hairpin H3. In the presence of target DNA, the hairpin structure of H1 was opened and catalytic hairpin assembly (CHA) was activated, and then a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were triggered and the product H1-H2 with sticky ends on both sides was formed. On the one side of H1-H2, the split two G-quadruplex sequences were close enough to form the intact G-quadruplex for the signal readout. At the same time, two sticky ends on the other side of H1-H2 hybridized with H3 and a new sticky end with the sequence same to the target DNA was exposed, which can immediately trigger the autocatalytic hairpin assembly reaction, and then the reaction rate of CHA was effectively accelerated and the colorimetric signal was significantly amplified. This ACHA signal amplified strategy has been successfully applied for the rapid and colorimetric nucleic acid detection.

Establishment of a Lateral Flow Dipstick Detection Method for Influenza A Virus Based on CRISPR/Cas12a System.

This study aimed to develop a rapid, visual PCR-CRISPR/Cas12-LFD method for detecting influenza A by utilizing the conserved region of the matrix protein gene. We crafted universal degradation primers and clustered regularly interspaced short palindromic repeats RNA (CRISPR RNA, crRNA) targeting the conserved matrix protein gene of the influenza virus (IFV), integrated with lateral flow dipstick (LFD) technology. This new PCR-CRISPR/Cas12-LFD approach was designed to determine its sensitivity and specificity through the analysis of various clinical samples collected in 2023. The developed nucleic acid assay for influenza A viruses (IAV) demonstrated a sensitivity of 10 copies/μL without cross-reactivity with other respiratory pathogens. Evaluation of 82 clinical samples showed high concordance with results from fluorescent Polymerase Chain Reaction (PCR), achieving a kappa value of 0.95. A highly sensitive and specific PCR-CRISPR/Cas12-LFD method has been successfully established for the detection of influenza A, offering a robust tool for its diagnosis and aiding in the prevention and control of this virus.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.

Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 101 copies·μL-1. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.

Simple and Multiplexed Detection of Nucleic Acid Targets Based on Fluorescent Ring Patterns and Deep Learning Analysis.

Simple diagnostic tests for nucleic acid targets can provide great advantages for applications such as rapid pathogen detection. Here, we developed a membrane assay for multiplexed detection of nucleic acid targets based on the visualization of two-dimensional fluorescent ring patterns. A droplet of the assay solution is applied to a cellulose nitrate membrane, and upon radial chromatographic flow and evaporation of the solvent, fluorescent patterns appear under UV irradiation. The target nucleic acid is isothermally amplified and is immediately hybridized with fluorescent oligonucleotide probes in a one-pot reaction. We established the fluorescent ring assay integrated with isothermal amplification (iFluor-RFA = isothermal fluorescent ring-based radial flow assay), and feasibility was tested using nucleic acid targets of the receptor binding domain (RBD) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2. We demonstrate that the iFluor-RFA method is capable of specific and sensitive detection in the subpicomole range, as well as multiplexed detection even in complex solutions. Furthermore, we applied deep learning analysis of the fluorescence images, showing that patterns could be classified as positive or negative and that quantitative amounts of the target could be predicted. The current technique, which is a membrane pattern-based nucleic acid assay combined with deep learning analysis, provides a novel approach in diagnostic platform development that can be versatilely applied for the rapid detection of infectious pathogens.

Tuning Hydrophobicity of Paper Substrates for Effective Colorimetricdetection of Glucose and Nucleic acids.

This study investigated the colorimetric response of standard glucose, serum glucose, and nucleic acid assays on various paper surfaces with different wettability, including hydrophilic, hydrophobic, and nearly superhydrophobic surfaces. Water contact angles (WCA) formed by water droplets on each surface were measured using ImageJ software. The hydrophilic surface showed no contact angle, while the hydrophobic and nearly superhydrophobic surfaces exhibited contact angles of 115.667° and 133.933°, respectively. The colorimetric sensitivity of the standard glucose assay was analyzed on these surfaces, revealing enhanced sensitivity on the nearly superhydrophobic surface due to the high molecular crowding effect owing to its non-wetting behavior and eventually confined reaction product at the sample loading zone. The hydrophobic nature of the surface restricts the spreading and diffusion of the reaction product, leading to a controlled and localized concentration of the assay product leading to moderate colorimetric intensity. On the other hand, the hydrophilic surface showed the least enhancement in colorimetric sensitivity; this is attributed to the high wettability of the hydrophilic surface causing the reaction product to spread extensively, resulting in a larger area of dispersion and consequently a lower colorimetric intensity. The measured limit of detection (LOD) for nucleic acid on nearly superhydrophobic surfaces was found to be 16.15ng/µL, which was almost four-fold lower than on hydrophilic surfaces (60.08ng/µL). Additionally, the LODs of standard glucose and clinical serum samples were two-fold lower on nearly superhydrophobic surfaces compared to hydrophilic surfaces. Our findings clearly highlight the promising potential of utilizing superhydrophobic surfaces to significantly enhance colorimetric sensitivity in paper-based diagnostic applications. This innovative approach holds promise for advancing point-of-care diagnostics and improving disease detection in resource-limited settings.

Gold nanoparticle-based strategies against SARS-CoV-2: A review

Abstract The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 has been an immense threat to global public health and has also had a negative socioeconomic impact worldwide. However, although the pandemic is now under control, it has demonstrated that society is unprepared to use analysis methods that are applicable to various types of viruses nor apply new therapies to prevent infections, considering the extensive time needed for vaccine development. The use of nanomaterial-based diagnostics and therapeutics can provide essential strategies for both virus detection and treatment. Gold nanoparticles (AuNPs) are the nanomaterials most commonly used to enhance virus detection because of their bioconjugation, high plasmon resonance, and excellent electrical, optical, and catalytic properties. The present review outlines the recent advances reported in the literature regarding using AuNPs for their antiviral activities with respiratory viruses, analysis techniques such as AuNP-assisted polymerase chain reaction, biosensors (electrochemical, piezoelectric, and optical), lateral flow analysis, nucleic acid assays, and gene and vaccine therapy. Finally, as a potential antiviral treatment, this review provides in vitro and in vivo toxicity results of AuNPs for respiratory viruses, as well as those related to their toxicity in humans, to evaluate their use as a future antiviral treatment.

Species-Level Monitoring of Key Bacteria in Fermentation Processes Using Single-Nucleotide Resolved Nucleic Acid Assays Based on CRISPR/Cas12.

Microorganisms can determine the flavor and quality of fermented food, such as Baijiu, which is produced via Daqu fermentation. Therefore, monitoring key microorganisms during fermentation is important for ensuring high-quality fermented food. Here, we report a single-nucleotide resolved nucleic acid assay based on the CRISPR/Cas12 system, enabling the quantification of Bacillus amyloliquefaciens, a key microorganism in Daqu fermentation at the species level. The assay employs an amplification-refractory mutation system derived from PCR to analyze minor genetic differences between different Bacillus species. The utilization of CRISPR/Cas12 further guaranties the specificity of identifying the PCR amplicon and enables the quantification of Bacillus amyloliquefaciens via end-measurement fluorescence. Compared to conventional qPCR, the assay allows for species-level detection of bacteria, thus enabling the precise detection of the Bacillus strain that yields high-level 2,3,5,6-tetramethylpyrazine. The assay promises the precise monitoring of bacterial growth and contribution to flavor during Daqu fermentation, thus facilitating fermented food quality control.

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, an mAb-based blocking enzyme-linked immunosorbent assay (bELISA) was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 4.89%, and 3.16%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with coronavirus disease 2019 (COVID-19)-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCE Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

Bettercallsal: better calling of Salmonella serotypes from enrichment cultures using shotgun metagenomic profiling and its application in an outbreak setting.

Most current Salmonella subtyping analyses rely on whole genome sequencing (WGS), which focuses on the high-resolution analysis of single genomes or multiple single genomes from the isolated colonies on microbiological agar plates. In this study, we introduce bioinformatics innovations for a metagenomic outbreak response workflow that accurately identifies multiple Salmonella serovars at the same time. bettercallsal is one of the first analysis tools to identify multiple Salmonella enterica serotypes from metagenomic or quasi-metagenomic datasets with high accuracy, allowing these isolate-independent methods to be incorporated into surveillance and root cause investigations. It was tested on an in silico benchmark dataset comprising 29 unique Salmonella serovars, 46 non-Salmonella bacterial genomes, and 10 viral genomes at varying read depths and on previously well-characterized and sequenced non-selective primary and selective enrichments of papaya and peach samples from separate outbreak investigations that resulted in the identification of multiple Salmonella serovars using traditional isolate culturing and WGS as well as nucleic acid assays. Analyses were also conducted on these datasets using a custom-built k-mer tool, SeqSero2, and Kallisto to compare serotype calling to bettercallsal. The in silico dataset analyzed with bettercallsal achieved the maximum precision, recall, and accuracy of 100, 83, and 94%, respectively. In the papaya outbreak samples, bettercallsal identified the presence of multiple serovars in agreement with the Luminex® xMAP assay results and also identified more serovars per sample, as evidenced by NCBI SNP clustering. In peach outbreak samples, bettercallsal identified two serovars in concordance with k-mer analysis and the Luminex xMAP assay. The genome hit reported by bettercallsal clustered with the chicken isolate genome, as reported by the FDA peach outbreak investigation from sequenced isolates (WGS). Overall, bettercallsal outperformed k-mer, Seqsero2, and Kallisto in identifying multiple serovars from enrichment cultures using shotgun metagenomic sequencing.

Enhancement of polyvinylpyrrolidone on antimicrobial activity and mechanism of Copper(II)-β-cyclodextrin

Copper-based materials are a cheap and efficient antibacterial agent that has received much attention for centuries. Copper(II)-β-cyclodextrin (Cu2-β-CD) can be applied at pH values in the range of 5.0–9.0, and its antibacterial effect remains unchanged. Herein, the antimicrobial activity of Cu2-β-CD were evaluated including the inhibition zone diameter, the minimum inhibitory concentration and the minimum bactericide concentration towards Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans). Interestingly, the antimicrobial activity of Cu2-β-CD was remarkably improved by adding polyvinylpyrrolidone (PVP), and the antimicrobial rates were increased from 96%, 62%, and 29% with single Cu2-β-CD to 100%, 100%, and 84% with Cu2-β-CD incorporated with 0.1 wt% PVP against E. coli, S. aureus, and C. albicans, respectively, whereas PVP had almost no antimicrobial effect. In addition, cell membrane integrity and cell wall integrity were severely destroyed by Cu2-β-CD/PVP according to the results of the permeability assay of cell membrane, the leakage assay of nucleic acids, protein and alkaline phosphatase, and scanning electron microscopy. Hence, this work presented a novel prospective and broad-spectrum antimicrobial system consisting of Cu2-β-CD and PVP.

A colloidal gold test strip based on catalytic hairpin assembly for the clinical detection of influenza a virus nucleic acid

Influenza A epidemics, which occur annually in varying degrees worldwide, is a global challenge to healthcare facilities owing to several limitations of the current detection methods. Therefore, the development of a rapid, convenient, and economical method for the early diagnosis of influenza A will aid clinical treatment and epidemic control. Currently, most of the commonly used clinical rapid tests utilize colloidal gold test strips that detect specific influenza virus antigens but are limited by low sensitivity. Therefore, this study combined catalytic hairpin assembly (CHA) with colloidal gold immunochromatographic assay (GICA) to develop a highly sensitive and visual CHA-GICA test strip. Clinical sample analysis revealed that the sensitivity of the assay was 81.8% and 74% under optimal (35 °C) and room temperature (25 °C) conditions, respectively. In conclusion, this study developed a rapid nucleic acid assay for detecting influenza A virus with high sensitivity and specificity, which can improve the clinical detection of influenza A.

Multi-Analyte Discriminated ECL via Photonic Crystal-Enhanced Selectivity.

Electrochemiluminescence (ECL) has numerous merits such as high sensitivity and specificity for the detection applications on pharmacy, food safety, immunoassay, disease diagnosis, environmental monitoring, nucleic acid assay, and clinical treatment. However, the insufficiency of ECL luminescent reagents is restricting their adoption on complex systems or multi-analyte detections. In this work, to improve the selectivity and discrimination of ECL detection with one or less luminescent reagent, we employed multi-stopband photonic crystals (PCs) to enhance assigned ECL. The discrimination of ECL was well investigated to establish the quantitative description with PC stopbands. The multi-stopband PC electrode can facilely achieve 10 antibiotics qualitative and quantitative analysis with 100% accuracy and 0.44 μM LOD in PBS buffer and human serum. The selectivity of ECL detection for multi-analytes can be improved via designed PC luminescence amplifications. The exploration on PC selectivity for ECL enhancement will promote the realistic application of the ECL technique and contribute to the facile and efficient optical platform for clinical or health monitoring.