Method For Detection Of Virus Research Articles

Circulation and Codetections of Influenza Virus, SARS-CoV-2, Respiratory Syncytial Virus, Rhinovirus, Adenovirus, Bocavirus, and Other Respiratory Viruses During 2022–2023 Season in Latvia

This retrospective study analysed the routine data obtained by multiplex real-time RT-qPCR methods for respiratory virus detection. A total of 4814 respiratory specimens collected during 1 September 2022–31 August 2023 were included in the study. A total of 38% of the specimens were positive for at least one target, with the incidence maximum (82%) for the small children (age group 0–4 years). The five dominant virus groups were rhinovirus (RV, 12%), influenza virus A (IAV, 7%), adenovirus (AdV, 6%), respiratory syncytial virus (RSV, 5%), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, 5%). The specimens with multi-detections represented 19% of the positives, unevenly distributed (n = 225, 56, 43, 24) among the age groups 0–4, 5–14, 15–64, and 65< years, respectively. The dominant virus groups in multi-positive specimens were RV (53%), AdV (43%), and bocavirus (BoV, 35%)—in mutual pairs as well as all three together—followed by RSV (21%), and IAV (15%). Our study focused on the specimens with codetections and provides an insight into the variety of the respiratory virus interactions in Latvia during the first year since pandemic-related social restriction measures were eased. The observations also emphasise the need to consider the differentiation between rhinoviruses and enteroviruses, especially for the youngest patients in the age group 0–4.

Bifunctional NiFe-LDH@AgCit nanocomposite-based immunosensor for electrochemical and colorimetric detection of SARS-CoV-2

The COVID-19 pandemic caused by SARS-CoV-2 has precipitated a global public health emergency, necessitating the urgent development of rapid and accurate virus detection methods. Compared with the current single-signal detection methods commonly used, dual-mode detection has inherent self-verification and self-correction functions and is expected to provide more reliable and interference-resistant detection results. In this work, a rapid approach for SARS-CoV-2 detection was developed using an electrochemical and colorimetric immunosensor based on a NiFe layered double hydroxide (LDH) loaded with silver citrate (NiFe-LDH@AgCit). The electrochemical sensor is capable of measuring the electrical signal of the transition from Ag to Ag+ in the presence of SARS-CoV-2. At the same time, the color reaction of 3,3′,5,5′-tetramethylbenzidine with ⋅OH can be used to build a colorimetric sensor. In electrochemical and colorimetric sensing systems, the linear detection ranges were 0.0005–10 and 0.001–10 ng mL−1, respectively. The corresponding limits of detection reached as low as 0.167 and 0.333 pg mL−1, respectively. In addition, the dual-signal sensor has been favorably used for the detection of SARS-CoV-2 in human serum, and the recovery ranges were 99.0 %–101.6 % and 100.6 %–104.5 %, respectively. In conclusion, the proposed dual-signal sensing technique offers novel insights for the prompt diagnosis of virus-related illnesses, which has wide application prospects.

Development and application of a quantitative real-time PCR method for detection of Decapod iridescent virus 1.

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/μL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1.

Evaluation of an Enzyme-Linked Magnetic Electrochemical Assay for Hepatitis a Virus Detection in Drinking and Vegetable Processing Water

Globally, waterborne viral infections significantly threaten public health. While current European Union regulations stipulate that drinking water must be devoid of harmful pathogens, they do not specifically address the presence of enteric viruses in water used for irrigation or food production. Traditional virus detection methods rely on molecular biology assays, requiring specialized personnel and laboratory facilities. Here, we describe an electrochemical sandwich enzyme-linked immunomagnetic assay (ELIME) for the detection of the hepatitis A virus (HAV) in water matrices. This method employed screen-printed electrodes as the sensing platform and utilized commercially available pre-activated magnetic beads to provide a robust foundation for the immunological reaction. The ELIME assay demonstrated exceptional analytical performance in only 185 min achieving a detection limit of 0.5 genomic copies per milliliter (g.c./mL) and exhibiting good reproducibility with a relative standard deviation (RSD) of 7% in HAV-spiked drinking and processing water samples. Compared with the real-time RT-qPCR method described in ISO 15216-1, the ELIME assay demonstrated higher sensitivity, although the overall linearity of the method was moderate. These analytical attributes highlight the potential of the ELIME assay as a rapid and viable alternative for HAV detection in water used for agriculture and food processing.

Microfluidic-free droplet digital RAA/RT-RAA assisted CRISPR/Cas12a system for the detection of DNA/RNA viruses

Digital PCR (dPCR) is the most sensitive method for virus detection by accurate quantifying the nucleic acids. However, the requirement of sophisticated and expensive instrumentations greatly limits its application in clinical diagnosis particularly in poorly resourced settings. Herein, we developed a simple digital assay based on polydisperse droplet digital recombinase-aided amplification/reverse transcription recombinase-aided amplification dependent CRISPR/Cas12a system (PddRaaCas/PddRRaaCas) for viral DNA/RNA detection with minimal instruments. This assay does not require any microfluidic chip for droplet generation and can be completed about 1 hour. We found that the PddRaaCas and PddRRaaCas enabled to detect 0.015 fM target DNA and 10 fM target RNA respectively. In addition, we applied this method to detect human papillomavirus type 18 (HPV18) and severe acute respiratory syndrome coronavirus (SARS-CoV-2) in clinical samples, which represented the detection of DNA virus and RNA virus respectively. The sensitivity and specificity were 100 % and 100 % for both DNA and RNA virus detection respectively. Taken together, these results highlight that PddRaaCas/PddRRaaCas is a sensitive, rapid, and easy-to-use strategy for the detection of DNA/RNA viruses, which has great potential application for the control of infectious diseases.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

Synergistic Role of the AuAg-Fe3O4 Nanoenzyme for Ultrasensitive Immunoassay of Dengue Virus.

A combination of magnetic and noble metal nanoparticles (NPs) has recently emerged as a potential substance for rapid and sensitive immunosorbent assays. However, to make the assay an alternative method for Enzyme-linked immunosorbent assay, the individual role of each nanoparticle must be explored properly. In this work, an immunoassay has been proposed using two antibody-conjugated iron oxide nanoparticles (Fe3O4NPs) and gold-silver bimetallic nanoparticles (AuAgNPs) to enhance the sensitivity of virus detection by colorimetric TMB/H2O2 signal amplification. A synergistic effect is monitored between Fe3O4NPs and AuAgNPs, which is explored for colorimetric virus detection. The sensor exploits the synergistic effect between the nanoparticles to successfully detect a wide range of dengue virus-like particle (DENV-LP) concentrations ranging from 10 to 100 pg/mL with a detection limit of up to 2.6 fg/mL. In the presence of a target DENV-LP, a sandwich-like structure is formed, which restricts the electron transfer and the associated synergistic effect between the nanoparticles, restricting the TMB oxidation process. Therefore, the synergistic effect is the key to the present work, which accounts for the enhanced rate of the enzymatic reaction on TMB and makes the current method of virus detection more sensitive and reliable compared to the others.

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA.

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

Improved passive sampling methods for wastewater to enable more sensitive detection of SARS-CoV-2 and its variants

Wastewater-based epidemiology (WBE) can be used as a part of a long-term strategy for detecting and responding rapidly to new outbreaks of infectious disease in the community. However, wastewater collected by grab samples may miss marker presence, and composite auto-sampling throughout a day is technically challenging and costly. Tampon swabs can be used as passive collectors of wastewater markers over hours, but recovery of the captured markers is a challenge. Our goal was to improve tampon elution methods for virus detection and variant analysis to increase the likelihood of detection near the Limit of Detection (LOD) and to potentially detect new or rare variants in a new outbreak. Counts of SARS-CoV-2 N1 and N2 markers in grab samples were compared to markers eluted from tampons that had been immersed in 3 sewersheds for 4–6 h during June to December 2023. We compared tampon elution methods that used different elution volumes, pressure, and amounts of Tween 20, evaluated after automated magnetic bead purification and RT-ddPCR of SARS-CoV-2 markers. Overall, method “SwabM2” in which tampons were eluted by high pressure squeeze in a 50 mL syringe after adding 2 mL of 0.5 X TE + 0.075 % Tween-20 yielded a median four-fold higher concentration of final purified SARS-CoV-2 markers than paired grab samples and significantly more than other tested tampon elution methods (p < 0.0001). Method SwabM2 was more likely to yield enough extracted nucleic acids for sequencing and also gave higher quality variant sequences than two other tampon elution methods. Variant analysis captured the Fall 2023 transition of variants from XBB to JN and “H” lineages. In summary, we demonstrated a tampon-based wastewater collecting and elution method that yielded higher counts, more detections near the LOD, and higher quality variant sequences compared to both grab samples and other tampon-based passive-collecting wastewater methods.

Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput sequencing

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

The Role of Oncogenic Viruses in Head and Neck Cancers: Epidemiology, Pathogenesis, and Advancements in Detection Methods.

Head and neck cancers (HNCs) constitute a wide range of malignancies originating from the epithelial lining of the upper aerodigestive tract, including the oral cavity, pharynx, larynx, nasal cavity, paranasal sinuses, and salivary glands. Although lymphomas affecting this region are not conventionally classified as HNCs, they may occur in lymph nodes or mucosa-associated lymphoid tissues within the head and neck. Oncogenic viruses play a crucial role in HNC onset. Human papillomavirus (HPV) is extensively studied for its association with oropharyngeal cancers; nevertheless, other oncogenic viruses also contribute to HNC development. This review provides an overview of the epidemiology, pathogenesis, and advancements in detection methods of oncogenic viruses associated with HNCs, recognizing HPV's well-established role while exploring additional viral connections. Notably, Epstein-Barr virus is linked to nasopharyngeal carcinoma and lymphomas. Human herpesvirus 8 is implicated in Kaposi's sarcoma, and Merkel cell polyomavirus is associated with subsets of HNCs. Additionally, hepatitis viruses are examined for their potential association with HNCs. Understanding the viral contributions in the head and neck area is critical for refining therapeutic approaches. This review underlines the interaction between viruses and malignancies in this region, highlighting the necessity for ongoing research to elucidate additional mechanisms and enhance clinical outcomes.

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.

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.

Label-Free Detection of Virus-like Particles with Surface-Enhanced Raman Spectroscopy through Analyte Localization and Polymer-Enabled Capture.

Virus detection is highly important; the last several years, since the onset of the SARS-CoV-2 pandemic, have highlighted a weakness in the field: the need for highly specialized and complex methodology for sensitive virus detection, which also manifests as sacrifices in limits of detection made to achieve simple and rapid sensing. Surface-enhanced Raman spectroscopy (SERS) has the potential to fill this gap, and two novel approaches to the development of a detection scheme are presented in this study. First, the physical entrapment of vesicular stomatitis virus (VSV) and additional virus-like particles through substrate design to localize virus analytes into SERS hotspots is explored. Then, the use of nonspecific linear polymers as affinity agents to facilitate polymer-enabled capture of the VSV for SERS detection is studied. Quantitative detection of the VSV is achieved down to 101 genetic copies per milliliter with an R2 of 0.987 using the optimized physical entrapment method. Physical entrapment of two more virus-like particles is demonstrated with electron microscopy, and distinctive SERS fingerprints are shown. This study shows great promise for the further exploration of label-free virus detection methods involving thoughtful substrate design and unconventional affinity agents.

SEQUENCING PROTOCOL AND BIOINFORMATICS PIPELINE FOR THE AVIAN INFLUENZA VIRUS

The avian influenza virus (AIV), traditionally confined to avian hosts, has recently been detected in various mammal species, raising significant concerns for both animal and public health, necessitating efficient and accurate methods for virus detection and characterization. This study presents a sequencing protocol combined with a comprehensive bioinformatics pipeline designed for the sequencing and analysis of AIV genomes. The presented streamlined approach encompasses whole genome PCR-amplification of the viral genome, enabling the genome characterization and detection of viral mutations with high precision. An amplicon-based MiniSeq sequencing workflow based on a set of PCR primers targeting all genome segments was developed. Three samples from H5 high pathogenic avian influenza (HPAI) outbreaks in Serbia were sequenced using the MiniSeq platform. The protocol involves optimized sample preparation, tailored specifically for AIV, library preparation and sequencing. This is complemented by a robust bioinformatics pipeline that includes quality control, read mapping, consensus genome generation, subtyping and pathotyping, as well as statistical sequencing data. The pipeline efficiently processes raw sequencing data, ensuring high-quality genome assemblies and accurate identification of viral strains. The protocol was used on AIV samples from various avian species, demonstrating its applicability and reliability. The results highlight the protocol’s capability to generate comprehensive genomic data, which is crucial for monitoring viral evolution and informing public health interventions. The described integrated approach offers a powerful tool for AIV surveillance and research, facilitating timely and informed decision-making in response to avian influenza outbreaks. This protocol can be readily adapted for use in various laboratory settings, contributing to global efforts in combating avian influenza and enhancing our understanding of its genomic landscape.

A Portable Fluorescent Lateral Flow Immunoassay Platform for Rapid Detection of FluA.

The spread of the FluA virus poses significant public health concerns worldwide. Fluorescent lateral flow immunoassay (LFIA) test strips have emerged as vital tools for the early detection and monitoring of influenza infections. However, existing quantitative virus-detection methods, particularly those utilizing smartphone-based sensing platforms, encounter accessibility challenges in resource-limited areas and among the elderly population. Despite their advantages in speed and portability, these platforms often lack user-friendliness for these demographics, impeding their widespread utilization. To address these challenges, this study proposes leveraging the optical pick-up unit (OPU) sourced from commercial optical drives as a readily available fluorescence excitation module for the quantitative detection of antibodies labeled with quantum-dot fluorescent microspheres. Additionally, we utilize miniaturized and high-performance optical components and 3D-printed parts, along with a customized control system, to develop an affordable point-of-care testing (POCT) device. Within the system, a stepping motor scans the test strip from the T-line to the C-line, enabling the calculation of the fluorescence-intensity ratio between the two lines. This simple yet effective design facilitates rapid and straightforward field or at-home testing for FluA. The proposed prototype platform demonstrates promising performance, achieving a limit of detection (LOD) of 2.91 ng/mL, a total detection time of no more than 15 min, and dimensions of 151 mm × 11.2 mm × 10.8 mm3. We believe that the proposed approach holds great potential for improving access to an accurate influenza diagnosis.

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/μL (blue light) and 1.9 × 103 copies/μL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

Identification of unique electromagnetic signatures from GLRaV-3 infected grapevine leaves in different stages of virus development.

Grapevine leafroll associated virus 3 (GLRaV-3) is one of the most common viruses that adversely affect grape production quality around the world. Grapevine virus is usually detected by polymerase chain reaction (PCR) tests, but the main diagnostic challenges with the PCR are uneven distribution of symptoms, low virus titers in early stage, and the tests are time consuming and expensive. In the absence of effective control and treatment for grapevine virus, the recommendation is to remove infected vines before they become a source of transmission and a rapid and early detection method for viruses is urgently needed. This study investigated how GLRaV-3 infection affects spectral behavior of grapevine leaves and the use of remote sensing tools to identify changes caused by the biotic stress. A Real-Time RT-qPCR test was performed to select representative vines with healthy and GLRaV-3 infection, and changes in electromagnetic (EM) spectrum of leaves were observed with a portable hyperspectral spectrometer and a multispectral sensor on a remotely piloted aircraft system (RPAS). The results of this study suggest that foliar EM reflectance may differ depending on the presence or absence of visible symptoms of infected leaves and different stages of virus infection progress could be indicated by changes in the relative rate of reflectance around 700 nm. This research also confirmed that the development of a narrow-band hyperspectral index for the detection of grapevine virus is feasible, further research is recommended in a much more comprehensive investigation including a combination of hyperspectral sensor and chlorophyll fluorescence sensors, interpolation of suspect wavebands into ratios or indices, larger sample sizes and a more controlled study environment.

Multiplex PCR methods for simultaneous detection of tomato brown rugose fruit virus, tomato spotted wilt virus and pepino mosaic virus

Multiplex <scp>PCR</scp> methods for simultaneous detection of tomato brown rugose fruit virus, tomato spotted wilt virus and pepino mosaic virus