Method For Detection Of Virus Research Articles

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.

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.

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

Validation of a next generation sequencing method for adventitious virus detection: Demonstration of sensitivity in multiple cell lines

In the past few years NGS has become the technology of choice to replace animal-based virus safety methods and this has been strengthened by the recent revision to the ICHQ5A virus safety chapter. Here we describe the validation of an NGS method using an agnostic analysis to detect and identify RNA virus and actively replicating DNA virus contaminants in cell banks. We report the results of the validation of each step in the sequencing process that established quality criteria to ensure consistent sequencing data. Furthermore, the validation of the analysis algorithm designed to identify virus specific sequences is described along with steps undertaken to ensure the integrity of the sequencing data from generation to analysis. Lastly, the validated sequencing and analysis systems were used to establish a limit of detection (LOD) for model viruses in cells that are commonly used in biomanufacturing. The LOD from these studies ranged from 1E+03 to 1E+04 genome copies and were dependent on the virus type with little variability between the different cell types. Thus, the validation of the NGS method for adventitious agent testing and the establishment of a general LOD for cell-based samples provides a suitable alternative to traditional virus detection methods.

Self-Assembly of Single-Virus SERS Hotspots for Highly Sensitive In Situ Detection of SARS-CoV-2 on Solid Surfaces.

Microbial surface transmission has aroused great attention since the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Developing a simple in situ detection method for viruses on solid surfaces is of great significance for timely public health surveillance. Taking advantage of the natural structure of SARS-CoV-2, we reported the assembly of Au@AgNPs on the surface of a single virus by the specific aptamer-spike protein interaction. Multiple hotspots can be created between the neighboring Au@AgNPs for the highly sensitive surface-enhanced Raman scattering (SERS) detection of SARS-CoV-2. Using two different aptamers labeled with Cy3 and Au@AgNPs, in situ SERS detection of pseudotyped SARS-CoV-2 (PSV) on packaging surfaces was achieved within 20 min, with a detection limit of 5.26 TCID50/mL. For the blind testing of 20 PSV-contaminated packaging samples, this SERS aptasensor had a sensitivity of 100% and an accuracy of 100%. This assay has been successfully applied to in situ detection of PSV on the surfaces of different packaging materials, suggesting its potential applicability.

Current Views About the Link between SARS-CoV-2 and the Liver: Friends or Foe?

The emergence of the novel coronavirus and the pandemic resulting from its spread have led to significant negative impacts on health, economy, relationships, and others. Particularly in the field of hospital care, the need for a greater number of patients has led to a breakdown of the system. Gastrointestinal manifestations are common in SARS-COV 2 patients, while 10% of those who are sick exhibit symptoms only from gastrointestinal without any manifestation on the part of the respiratory tract. The main manifestations are nausea, vomiting, diarrhoea, and anorexia. It is also interesting to note that biochemical liver disorder is a frequent finding and is associated with a worse prognosis and higher probability admission to intensive care. It was also observed that RNA from the virus was found in the stool several days after the tests came back negative pulmonary secretions, while rectal swab virus detection methods were used with a lower but comparable sensitivity to that of nasal swabs. Gastrointestinal symptoms in SARS-COV 2 infection are common and their search should be part of the initial diagnosis approach and have a connection with the gut microbiota dysbiosis and this can lead to an alteration of the gut/liver axis.

Optimization of Amplicon-Based Sequencing for Large-Scale Diagnostics of Known Pome Viruses and Viroids

The implementation of reliable methods for the simultaneous detection of multiple viruses is challenging in plant viral diagnosis. In the present study, we report the development and effectiveness of an optimized version of a robust multiplexed PCR (HiPlex) followed by simultaneous targeted amplicon sequencing to detect 14 viruses and 5 viroids of pome fruit trees. We used amplicon sequencing data from 1,400 pome fruit tree samples previously diagnosed with the HiPlex v.1.0, which included 290 primer pairs for pome viruses. The analysis enabled the identification of highly efficient and specific primer pairs to be assembled in a new HiPlex (dubbed HiPlex v.2.0). Additionally, newly designed primers were implemented for 30% of targeted viruses and viroids. Overall, 116 primer pairs were assembled into the HiPlex v.2.0, resulting in a reduction of the number of primers and multiplex PCR reactions needed by 60 and 50%, respectively. The new HiPlex also includes two nepoviruses, tomato ringspot virus and tobacco ringspot virus, showing flexibility at augmenting and expanding the number of targeted diagnosed viruses using this approach. This optimized set of primers increased the cost-effectiveness and sensitivity while reducing background amplification. Comparable results for virus and viroid detection were obtained using the HiPlex v.2.0 and individual RT-qPCRs, even at a 10,000-fold dilution, demonstrating similar sensitivity to standard PCR-based diagnostics. This new HiPlex-based amplicon sequencing represents a powerful, sensitive, and low-cost tool for large-scale screening of pome fruit trees for all reported viruses and viroids infecting these crops. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Infectious Keratitis.

To determine the advantages of next-generation metagenomic sequencing (mNGS) technology in the diagnosis and treatment of infectious keratitis (IK). A total of 287 patients with IK admitted to the Department of Ophthalmology of Nanjing First Hospital between August 2018 and December 2022 were analyzed retrospectively, and the pathogenic causes, etiological characteristics, detection, treatment methods, and efficacy were summarized. Trauma and foreign matter were the most common causes of IK (144 patients, 50.2%). Of the 287 patients, 228 (79.4%) were diagnosed with a specific etiology, including 110 (48.2%) fungal infections, 44 (19.3%) viral infections, 42 (18.4%) mixed infections, and 30 (13.2%) bacterial infections. Filamentous fungi represented by Fusarium and Aspergillus were the most common, followed by bacteria such as Pseudomonas aeruginosa, Streptococcus pneumoniae, viruses (Herpes Simplex Virus/Varicella-Zoster Virus), and parasites. The positivity rates of secretion culture, corneal laser confocal microscopy (CM), mNGS, and pathological sections were 47.3% (133/281), 45.3% (111/245), 83.9% (104/124), and 19.3% (40/207), respectively. The positivity rate of mNGS for bacteria and viruses was higher than that of the other methods, and the positivity rate for fungi was the same as that for CM. As a result, 214 cases (74.6%) were cured, 51 cases (17.8%) improved, 8 cases (2.8%) did not heal, ocular content enucleation was performed in 14 cases (4.9%), and the overall efficacy rate was 92.3%. Trauma and foreign matter are the main causes of IK. The mNGS technology is an efficient and comprehensive detection method for viruses and bacteria, especially for mixed infections.

Bis-tridentate Iridium(III) Complex with the N-Heterocyclic Carbene Ligand as a Novel Efficient Electrochemiluminescence Emitter for the Sandwich Immunoassay of the HHV-6A Virus.

Human herpesvirus type 6A (HHV-6A) can cause a series of immune and neurological diseases, and the establishment of a sensitive biosensor for the rapid detection of HHV-6A is of great significance for public health and safety. Herein, a bis-tridentate iridium complex (BisLT-Ir-NHC) comprising the N-heterocyclic carbene (NHC) ligand as a novel kind of efficient ECL luminophore has been unprecedently reported. Based on its excellent ECL properties, a new sensitive ECL-based sandwich immunosensor to detect the HHV-6A virus was successfully constructed by encapsulating BisLT-Ir-NHC into silica nanoparticles and embellishing ECL sensing interface with MXene@Au-CS. Notably, the immunosensor illustrated in this work not only had a wide linear range of 102 to 107 cps/μL but also showed outstanding recoveries (98.33-105.11%) in real human serum with an RSD of 0.85-3.56%. Undoubtedly, these results demonstrated the significant potential of the bis-tridentate iridium(III) complex containing an NHC ligand in developing ECL-based sensitive analytical methods for virus detection and exploring novel kinds of efficient iridium-based ECL luminophores in the future.

Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and Odontoglossum ringspot virus.

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world's most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 104 and 103 copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/μL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.