Rapid Detection Of Viruses Research Articles

Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.

Recent Advances in DNA Nanotechnology-Based Sensing Platforms for Rapid Virus Detection.

Several major viral pandemics in history have significantly impacted the public health of human beings. The COVID-19 pandemic has further underscored the critical need for early detection and screening of infected individuals. However, current detection techniques are confronted with deficiencies in sensitivity and accuracy, restricting the capability of detecting trace amounts of viruses in human bodies and in the environments. The advent of DNA nanotechnology has opened up a feasible solution for rapid and sensitive virus determination. By harnessing the designability and addressability of DNA nanostructures, a range of rapid virus sensing platforms have been proposed. This review overviewed the recent progress, application, and prospect of DNA nanotechnology-based rapid virus detection platforms. Furthermore, the challenges and developmental prospects in this field were discussed.

Progress and Outlooks in Designing Photonic Biosensor for Virus Detection

AbstractThe recent outbreak of coronavirus disease 2019 (COVID‐19) highlights the critical need for rapid, sensitive, and accurate virus detection methods to prevent and manage pandemics. Among the available sensing methods, photonic biosensors have emerged as a forefront technology, characterized by their high sensitivity, minimal analyte requirements, and suitability for miniaturization, making them ideal for point‐of‐care applications in virus detection. This review comprehensively summarizes the recent progress of photonic biosensor technologies, focusing on wavelength shift and luminescence‐based mechanisms. Their operational principles, general configurations, and the challenges associated with these technologies are looked into. An overview of the material developments used in photonic biosensors, encompassing organic, inorganic, and hybrid composite‐based materials is further presented. The discussion extends to surface functionalization using biorecognition elements, including DNA/RNA, aptamers, and antibodies, to craft the specificity of the photonic biosensors for viruses. Ultimately, the importance of a multidisciplinary approach is emphasized in developing new materials architecture, biological receptors, and modifications to photonic methods, aiming to realize better biosensors for virus detection with ultra‐high sensitivity, rapid response, and excellent selectivity.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

Rapid, sensitive, and visual detection of pseudorabies virus with an RPA-CRISPR/EsCas13d-based dual-readout portable platform

Pseudorabies viruses (PRV) pose a major threat to the global pig industry and public health. Rapid, intuitive, affordable, and accurate diagnostic testing is critical for controlling and eradicating infectious diseases. In this study, a portable detection platform based on RPA-CRISPR/EsCas13d was developed. The platform exhibits high sensitivity (1 copy/μL), good specificity, and no cross-reactivity with common pathogens. The platform uses rapid preamplification technology to provide visualization results (lateral flow assays or visual fluorescence) within 1 h. Fifty pig samples (including tissues, oral fluids, and serum) were tested using this platform and real-time quantitative polymerase chain reaction (qPCR), showing 34.0 % (17 of 50) PRV positivity with the portable CRISPR/EsCas13d dual-readout platform, consistent with the qPCR results. These results highlight the stability, sensitivity, efficiency, and low equipment requirements of the portable platform. Additionally, a novel point-of-care test is being developed for clinical use in remote rural and resource-limited areas, which could be a prospective measure for monitoring the progression of pseudorabies and other infectious diseases worldwide.

Evaluation of an Automated Ultrafiltration System for Concentrating a Range of Viruses from Saline Waters.

Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed.

Rapid Differential Detection of Wild-Type Classical Swine Fever Virus and Hog Cholera Lapinized Virus Vaccines by TaqMan MGB-Based Dual One-Step Real-Time RT-PCR.

To establish a rapid real-time RT-PCR method for differentiating wild-type classical swine fever virus (CSFV) strains from vaccine strains (HCLV), we designed a universal primer targeting the NS3 gene to detect wild-type CSFV strains and vaccine strains simultaneously, and two TaqMan-MGB probes were designed to differentiate between wild-type and vaccine strains. After optimizing the RT-qPCR conditions, a rapid dual TaqMan-MGB RT-qPCR method for the detection and identification of CSFV and HCLV was developed. The results showed that method could specifically detect CSFV and HCLV with no cross-reactivity with other swine pathogens. The analytic sensitivity for the NS3 gene of CSFV and HCLV were 1.67 × 101 copies/μL, respectively. For precision testing, the repeatability and reproducibility of the test was less than 2%. This method was successfully used for the rapid detection of 193 biological samples collected from CSFV-vaccinated pigs. This fast and accurate detection technology can be used for the detection of CSFV and is suitable for differentiating between wild-type CSFV strains and vaccine strains.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I-V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I-V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Novel triplex nucleic acid lateral flow immunoassay for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus

We report the development of a triplex nucleic acid lateral flow immunoassay (NALFIA) for the detection of the genomes of Nipah virus (NiV), Middle East respiratory syndrome coronavirus (MERS-CoV) and Reston ebolavirus (REBOV), which are intended for screening bats as well as other hosts and reservoirs of these three viruses. Our triplex NALFIA is a two-step assay format: the target nucleic acid in the sample is first amplified using tagged primers, and the tagged dsDNA amplicons are captured by antibodies immobilized on the NALFIA device, resulting in signal development from the binding of a streptavidin-colloidal gold conjugate to a biotin tag on the captured amplicons. Triplex amplification of the N gene of NiV, the UpE gene of MERS-CoV, and the Vp40 gene of REBOV was optimized, and three compatible combinations of hapten labels and antibodies were identified for end point detection. The lowest RNA copy numbers detected by the triplex NALFIA were 8.21e4 for the NiV N target, 7.09e1 for the MERS-CoV UpE target, and 1.83e4 for the REBOV Vp40 target. Using simulated samples, the sensitivity and specificity for MERS-CoV and REBOV targets were estimated to be 100%, while the sensitivity and specificity for the NiV target were 91% and 93.3%, respectively. The compliance rate between triplex NALFIA and real-time RT‒PCR was 92% for the NiV N target and 100% for the MERS-CoV UpE and REBOV Vp40 targets.

Bismuthene - Tetrahedral DNA nanobioconjugate for virus detection

In this work, we present an electrochemical sensor for fast, low-cost, and easy detection of the SARS-CoV-2 spike protein in infected patients. The sensor is based on a selected combination of nanomaterials with a specific purpose. A bioconjugate formed by Few-layer bismuthene nanosheets (FLB) and tetrahedral DNA nanostructures (TDNs) is immobilized on Carbon Screen-Printed Electrodes (CSPE). The TDNs contain on the top vertex an aptamer that specifically binds to the SARS-CoV-2 spike protein, and a thiol group at the three basal vertices to anchor to the FLB. The TDNs are also marked with a redox indicator, Azure A (AA), which allows the direct detection of SARS-CoV-2 spike protein through changes in the current intensity of its electrolysis before and after the biorecognition reaction. The developed sensor can detect SARS-CoV-2 spike protein with a detection limit of 1.74 fg mL−1 directly in nasopharyngeal swab human samples. Therefore, this study offers a new strategy for rapid virus detection since it is versatile enough for different viruses and pathogens.

Molecular Diagnostic Strategies Used for the Detection of Microbial Pathogens in Human Beings: A Review

Background: Viral diseases pose a serious health hazard to human population, worldwide. A perfect illustration of how a viral infection could pose a serious threat to public health and economic sectors is the current COVID-19 outbreak brought on by SARS-CoV-2 in 2019. Consequently, obtaining a prompt and accurate diagnosis is the first step in treating infections. For effective treatment, epidemic control, and prevention, early and precise identification of microbial presence in patient samples is essential. Methods: This study lists some of the molecular and immunological diagnostic methods that can be used to find infections in human beings. Rapid viral detection in patient samples is possible by the use of molecular diagnostic techniques. These techniques are also reasonably cheap, quite sensitive, and very targeted. Infections in human beings have been detected and the epidemiology of these illnesses has been widely studied using immunologically based methods. Results: In clinical samples, these methods can identify viral antigens or antiviral antibodies. Many commercially accessible molecular and immunological diagnostic kits make it easier to employ these techniques in most clinical laboratories around the world. Conclusion: This review offers a new perspective on molecular techniques employed in the application of the clinical diagnostics of microbes.

A Portable, Integrated, Sample-In Result-Out Nucleic Acid Diagnostic Device for Rapid and Sensitive Chikungunya Virus Detection.

Chikungunya virus, a mosquito-borne virus that causes epidemics, is often misdiagnosed due to symptom similarities with other arboviruses. Here, a portable and integrated nucleic acid-based diagnostic device, which combines reverse transcription-loop-mediated isothermal amplification and lateral-flow detection, was developed. The device is simple to use, precise, equipment-free, and highly sensitive, enabling rapid chikungunya virus identification. The result can be obtained by the naked eye within 40 min. The assay can effectively distinguish chikungunya virus from dengue virus, Japanese encephalitis virus, Zika virus, and yellow fever virus with high specificity and sensitivity as low as 598.46 copies mL-1. It has many benefits for the community screening and monitoring of chikungunya virus in resource-limited areas because of its effectiveness and simplicity. The platform has great potential for the rapid nucleic acid detection of other viruses.

Rapid detection of grapevine berry inner necrosis virus using a simple reverse transcription loop-mediated amplification method

To develop a simple method for the detection of grapevine berry inner necrosis (GINV) that does not depend on expensive instruments, a reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay was developed. A set of primers was used in RT-LAMP to specifically detect GINV and different GINV variants belonging to groups 1, 2 and 3. The sensitivity of the RT-LAMP was about 100 times greater than that of conventional RT-PCR. Furthermore, a rapid “pin-prick” assay without RNA extraction was developed to detect GINV in RT-LAMP. The assay performed well for the detection of GINV from different parts of field grapevine plants and micropropagated grapevine plantlets, and had a higher detection rate of GINV than RT-PCR in the tested grapevine samples.

A Wax Interface-Enabled One-Pot Multiplexed Nucleic Acid Testing Platform for Rapid and Sensitive Detection of Viruses and Variants.

High-quality, low-cost, and rapid detection is essential for the society to reopen the economy during the critical period of transition from Coronavirus Disease 2019 (COVID-19) pandemic response to pandemic control. In addition to performing sustainable and target-driven tracking of SARS-CoV-2, conducting comprehensive surveillance of variants and multiple respiratory pathogens is also critical due to the frequency of reinfections, mutation immune escape, and the growing prevalence of the cocirculation of multiple viruses. By utilizing a 0.05 cents wax interface, a Stable Interface assisted Multiplex Pathogenesis Locating Estimation in Onepot (SIMPLEone) using nested RPA and CRISPR/Cas12a enzymatic reporting system is successfully developed. This smartphone-based SIMPLEone system achieves highly sensitive one-pot detection of SARS-CoV-2 and its variants, or multiple respiratory viruses, in 40 min. A total of 89 clinical samples, 14 environmental samples, and 20 cat swab samples are analyzed by SIMPLEone, demonstrating its excellent sensitivity (3-6 copies/reaction for non-extraction detection of swab and 100-150 copies/mL for RNA extraction-based assay), accuracy (>97.7%), and specificity (100%). Furthermore, a high percentage (44.2%) of co-infection cases are detected in SARS-CoV-2-infected patients using SIMPLEone's multiplex detection capability.

Rational Design of 3D Polymer Corona Interfaces of Single-Walled Carbon Nanotubes for Receptor-Free Virus Recognition.

Facing the escalating threat of viruses worldwide, the development of efficient sensor elements for rapid virus detection has never been more critical. Traditional point-of-care (POC) sensors struggle due to their reliance on fragile biological receptors and limited adaptability to viral strains. In this study, we introduce a nanosensor design for receptor-free virus recognitions using near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) functionalized with a poly(ethylene glycol) (PEG)-phospholipid (PEG-lipid) array. Three-dimensional (3D) corona interfaces of the nanosensor array enable selective and sensitive detection of diverse viruses, including Ebola, Lassa, H3N2, H1N1, Middle East respiratory syndrome (MERS), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and SARS-CoV-2, even without any biological receptors. The PEG-lipid components, designed considering chain length, fatty acid saturation, molecular weight, and end-group moieties, allow for precise quantification of viral recognition abilities. High-throughput automated screening of the array demonstrates how the physicochemical properties of the PEG-lipid/SWCNT 3D corona interfaces correlate with viral detection efficiency. Utilizing molecular dynamics and AutoDock simulations, we investigated the impact of PEG-lipid components on 3D corona interface formation, such as surface coverage and hydrodynamic radius and specific molecular interactions based on chemical potentials. Our findings not only enhance detection specificity across various antigens but also accelerate the development of sensor materials for promptly identifying and responding to emerging antigen threats.

Loop-Mediated Isothermal Amplification and Lateral Flow Immunochromatography Technology for Rapid Diagnosis of Influenza A/B.

Influenza viruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Rapid detection of influenza viruses is essential for accurate diagnosis and the initiation of appropriate treatment. We developed a loop-mediated isothermal amplification and lateral flow assay (LAMP-LFA) capable of simultaneously detecting influenza A and influenza B. Primer sets for influenza A and influenza B were designed to target conserved regions of segment 7 and the nucleoprotein gene, respectively. Optimized through various primer set ratios, the assay operated at 62 °C for 30 min. For a total of 243 (85 influenza A positive, 58 influenza B positive and 100 negative) nasopharyngeal swab samples, the performance of the influenza A/B multiplex LAMP-LFA was compared with that of the commercial AllplexTM Respiratory Panel 1 assay (Seegene, Seoul, Korea). The influenza A/B multiplex LAMP-LFA demonstrated a specificity of 98% for the non-infected clinical samples, along with sensitivities of 94.1% for the influenza A clinical samples and 96.6% for the influenza B clinical samples, respectively. The influenza A/B multiplex LAMP-LFA showed high sensitivity and specificity, indicating that it is reliable for use in a low-resource environment.

Rapid Detection and Quick Characterization of African Swine Fever Virus Using the VolTRAX Automated Library Preparation Platform.

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease affecting domestic and wild swine. The current ASFV pandemic strain has a high mortality rate, severely impacting pig production and, for countries suffering outbreaks, preventing the export of their pig products for international trade. Early detection and diagnosis of ASFV is necessary to control new outbreaks before the disease spreads rapidly. One of the rate-limiting steps to identify ASFV by next-generation sequencing platforms is library preparation. Here, we investigated the capability of the Oxford Nanopore Technologies' VolTRAX platform for automated DNA library preparation with downstream sequencing on Nanopore sequencing platforms as a proof-of-concept study to rapidly identify the strain of ASFV. Within minutes, DNA libraries prepared using VolTRAX generated near-full genome sequences of ASFV. Thus, our data highlight the use of the VolTRAX as a platform for automated library preparation, coupled with sequencing on the MinION Mk1C for field sequencing or GridION within a laboratory setting. These results suggest a proof-of-concept study that VolTRAX is an effective tool for library preparation that can be used for the rapid and real-time detection of ASFV.

Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Development of recombinase amplification assays for the rapid detection of infectious myonecrosis virus

Infectious myonecrosis virus (IMNV) has affected shrimp farming in many countries, such as northeastern Brazil and southeast Asia, and poses a serious threat to the global shrimp industry. Reverse transcription enzymatic recombinant amplification technology (RT-ERA) is a rapid DNA amplification assay with high specificity in isothermal conditions and has been widely applied to the pathogen’s detection. In this study, two novel ERA assays of IMNV, real-time RT-ERA and an RT-ERA combined with lateral flow dipsticks assay (RT-ERA-LFD), were developed and evaluated. The real-time RT-ERA assay could be carried out at 38–42 °C and had the highest end-point fluorescence value and the smallest Ct value at 41 °C. The brightness and width of the detection line were at a maximum at 39 °C and 30 min, and these conditions were selected in RT-ERA-LFD. Both real-time RT-ERA and RT-ERA-LFD produced positive results with IMNV standard plasmids only and showed no cross-reaction with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease (VpAHPND); white spot syndrome virus (WSSV); infectious hypodermal and hematopoietic necrosis virus (IHHNV); or Ecytonucleospora hepatopenaei (EHP). Meanwhile, we compared the sensitivities of nested RT-PCR, real-time RT-PCR, real-time RT-ERA, and RT-ERA-LFD. The sensitivities of real-time RT-ERA and RT-ERA-LFD were both 101 copies/μL. The detection sensitivities of nested RT-PCR and real-time RT-PCR were 100 and 102 copies/μL, respectively. As a result, two ERA assays were determined to be specific, sensitive, and economical methods for the on-site diagnosis of IMNV infection, showing great potential for the control of IMNV infections.