Advances in biomacromolecule-functionalized magnetic particles for phytopathogen detection.

Due to the increasing crop losses caused by common and newly emerging phytopathogens, there is a pressing need for the development of rapid and reliable methods for phytopathogen detection and analysis. Leveraging advancements in biochemical engineering technologies and nanomaterial sciences, researchers have put considerable efforts on utilizing biofunctionalized magnetic micro- and nanoparticles (MPs) to develop rapid and reliable systems for phytopathogen detection. MPs facilitate the rapid, high-throughput analysis and in-field applications, while the biomacromolecules, which play key roles in the biorecognitions, interactions and signal amplification, determine the specificity, sensitivity, reliability, and portability of pathogen detection systems. The integration of MPs and biomacromolecules provides dimensionality- and composition-dependent properties, representing a novel approach to develop phytopathogen detection systems. In this review, we summarize and discuss the general properties, synthesis and characterization of MPs, and focus on biomacromolecule-functionalized MPs as well as their representative applications for phytopathogen detection and analysis reported over the past decade. Extensively studied bioreceptors, such as antibodies, phages and phage proteins, nucleic acids, and glycans that are involved in the recognitions and interactions, are covered and discussed. Additionally, the integration of MPs-based detection system with portable microfluidic devices to facilitate their in-field applications is also discussed. Overall, this review focuses on biomacromolecule-functionalized MPs and their applications for phytopathogen detection, aiming to highlight their potential in developing advanced biosensing systems for effective plant protection.

Cable Eccentricity Detection Method Based on Magnetic Field.

Amid the rapid advancement of electronic information technology, the need for cable eccentricity measurement in the industry is increasing both in China and across the globe. Current detection methods have several flaws, including high costs, insufficient accuracy, and instability. In this paper, we introduce a magnetic field-based detection method for cable eccentricity that provides high precision and cost-effectiveness. We position three pairs of magnetic field-collection modules in a circular array to gather magnetic flux density information induced by the electrified cable. We apply the law of electromagnetic induction to calculate the cable eccentricity. Our method is non-contact, preserving the cable's integrity. Our method outperforms traditional detection methods, not only in achieving greater accuracy and stability but also in significantly lowering the detection cost. Simulations and experiments show that our method's error rate under specified conditions is 0~4%, with a maximum standard deviation of 0.11, confirming its precision and stability in detecting cable eccentricity. The effectiveness of our method is influenced by two factors: lift-off value and loading current intensity. Our method presents a novel concept and a dependable strategy for the progress of cable eccentricity-detection technology.

Near-field detection and peak frequency metric for substrate and activation mapping of ventricular tachycardias in two- and three-dimensional circuits.

AIMS: Successful ventricular arrhythmia (VA) ablation requires identification of functionally critical sites during contact mapping. Estimation of the peak frequency (PF) component of the electrogram (EGM) may improve correct near-field (NF) annotation to identify circuit segments on the mapped surface. In turn, assessment of NF and far-field (FF) EGMs may delineate the three-dimensional path of a ventricular tachycardia (VT) circuit. METHODS AND RESULTS: A proprietary NF detection algorithm was applied retrospectively to scar-related re-entry VT maps and compared with manually reviewed maps employing first deflection (FDcorr) for VT activation maps and last deflection (LD) for substrate maps. Ventricular tachycardia isthmus location and characteristics mapped with FDcorr vs. NF were compared. Omnipolar low-voltage areas, late activating areas, and deceleration zones (DZ) in LD vs. NF substrate maps were compared. On substrate maps, PF estimation was compared between isthmus and bystander sites. Activation mapping with entrainment and/or VT termination with radiofrequency (RF) ablation confirmed critical sites. Eighteen patients with high-density VT activation and substrate maps (55.6% ischaemic) were included. Near-field detection correctly located critical parts of the circuit in 77.7% of the cases compared with manually reviewed VT maps as reference. In substrate maps, NF detection identified deceleration zones in 88.8% of cases, which overlapped with FDcorr VT isthmus in 72.2% compared with 83.3% overlap of DZ assessed by LD. Applied to substrate maps, PF as a stand-alone feature did not differentiate VT isthmus sites from low-voltage bystander sites. Omnipolar voltage was significantly higher at isthmus sites with longer EGM durations compared with low-voltage bystander sites. CONCLUSION: The NF algorithm may enable rapid high-density activation mapping of VT circuits in the NF of the mapped surface. Integrated assessment and combined analysis of NF and FF EGM-components could support characterization of three-dimensional VT circuits with intramural segments. For scar-related substrate mapping, PF as a stand-alone EGM feature did not enable the differentiation of functionally critical sites of the dominant VT from low-voltage bystander sites in this cohort.

Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/µl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

Three-dimensional electrochemical-magnetic-thermal coupling model for lithium-ion batteries and its application in battery health monitoring and fault diagnosis.

Storage batteries with elevated energy density, superior safety and economic costs continues to escalate. Batteries can pose safety hazards due to internal short circuits, open circuits and other malfunctions during usage, hence real-time surveillance and error diagnosis of the battery's operational state is imperative. In this paper, a three-dimensional model of electrochemical-magnetic field-thermal coupling is formulated with lithium-ion pouch cells as the research focus, and the spatial distribution pattern of the physical field such as magnetic field and temperature when the battery is operational is acquired. Furthermore, this manuscript also investigates the diagnostic methodology for defective batteries with internal short circuits and fissures, that is, the operational state of the battery is evaluated and diagnosed by the distribution of the magnetic field surrounding the battery. To substantiate the method's practical viability, the present study extends its examination to the 18650-battery pack. We obtained the magnetic field images of the normal operation of the battery pack and the failure state of some batteries and analyzed the relationship between the magnetic field distribution characteristics and the performance of the battery pack, providing a new method for the health monitoring and fault diagnosis of the battery pack. This non-contact method incurs no damage to the battery, concurrently exhibiting elevated sensitivity and extremely rapid response time. Meanwhile, it provides an effective means for non-destructive research on the batteries and can be applied to areas such as battery safety screening and non-destructive testing. This research not only helps to facilitate our understanding of the battery's operating mechanism, but also provides robust support for safe operation and optimal battery design.

Portable one-step effervescence tablet-based microextraction combined with smartphone digital image colorimetry: Toward field and rapid detection of trace nickel ion.

In this study, the one-step switchable hydrophilic solvent (SHS)-based effervescence tablet microextraction (ETME) was coupled with smartphone digital image colorimetry (SDIC) for the field detection of nickel ion (Ni2+) for the first time. Both extractant and CO2 were generated in situ when the novel SHS-based effervescence tablet was placed in the sample solution. The complexant 1-(2-pyridinylazo)-2-naphthaleno (PAN) dissolved from the effervescence tablet to form a stable complex with Ni2+, and the extractant was uniformly dispersed in the sample solution under the action of CO2 and fully in contact with Ni-PAN, which enabled efficient extraction of Ni2+. The color changes of the extraction phase were captured by smartphone, then a quantitative relationship between the concentrations of Ni2+ and color intensity of images captured using a smartphone was established by customized applet WASDIC, which realized quantitative analysis of Ni2+ in different samples. Under optimal conditions, the enhancement factor (EF) of the proposed method was 65.1, the limit of detection (LOD) and limit of quantification (LOQ) were 1.69 and 5.64 µg L-1, respectively. The developed method was successfully applied to the detection of trace Ni2+ in the environmental samples and natural medicines. And the applicability of the method for use in field analysis was validated.

Rapid authentication of characteristic milk powders by recombinase polymerase amplification assays.

The authentication of dairy species has great significance for food safety. This study focused on a more rapid method for identifying major dairy species, and specific recombinase polymerase amplification (RPA)-based assays for cattle, goat, sheep, camel and donkey were developed. Through the developed RPA-based assays, goats and sheep could be simultaneously identified and bovine families could be differentiated. The performances of the RPA assays were validated using 37 milk powder samples, of which 16.2% (6/37) were suspected of being adulterated and 24.3% (9/37) were potentially at risk of being wrongly identified as adulteration. The effectiveness of the developed assays for crude DNA detection was also validated by a rapid nucleic acid extraction kit, and results showed that the presence of large amounts of protein and fat did not affect the qualitative results. Therefore, these assays could combine with the rapid nucleic acids extraction methods for being used in field detection.

In-field detection and characterization of B/Victoria lineage deletion variant viruses causing early influenza activity and an outbreak in Louisiana, 2019.

Background: In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. Method: As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real-time RT-PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. Results: The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real-time RT-PCR). Conclusion: This work underscores the value of portable platforms for rapid, onsite pathogen characterization.

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020-2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.

Vacuum Oven Drying: A Cost-Effective Way of Producing Field-Deployable Reagents for In-house Real-Time PCR Methods.

The polymerase chain reaction (PCR), is a widely used, sensitive and reliable method for detecting pathogens. However, technical limitations may restrict its use outside sophisticated laboratories, e.g. for detecting pathogens at the site of a disease outbreak. In this study, real-time PCR reagents specific to four bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and to the Influenza A virus were dried using a vacuum oven drying method. The performance of the dried reagents stored at different temperatures, was monitored using both a standard-size and a portable real-time PCR instrument. The vacuum oven dried real-time PCR reagents were stable and retained the sensitivity for at least 14 months when stored in a refrigerator (+ 4 °C). When stored at room temperature, DNA assays remained stable for at least 10 weeks and Influenza A RNA assay for 3 weeks. These results demonstrate the feasibility of vacuum oven dried real-time PCR reagents and a portable thermocycler for the rapid and reliable detection of pathogens. The drying protocol presented here is cost-effective and easy to use, and could be applied to real-time PCR methods specific to other pathogens as well. In addition, this in-house drying protocol reduces reliance on commercial PCR tests during a time of shortage, such as that experienced during the Corovirus disease (COVID-19) crisis.

Development of a reverse transcription loop-mediated isothermal amplification based clustered regularly interspaced short palindromic repeats Cas12a assay for duck Tembusu virus.

Duck Tembusu virus (DTMUV) is an emerging pathogen that poses a serious threat to the duck industry in China. Currently, polymerase chain reaction (PCR), quantitative PCR (qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are commonly used for DTMUV detection. However, these methods require complex steps and special equipment and easily cause false-positive results. Therefore, we urgently need to establish a simple, sensitive and specific method for the clinical field detection of DTMUV. In this study, we developed an RT-LAMP-based CRISPR-Cas12a assay targeting the C gene to detect DTMUV with a limited detection of 3 copies/µL. This assay was specific for DTMUV without cross-reaction with other common avian viruses and only required some simple pieces of equipment, such as a thermostat water bath and blue/UV light transilluminator. Furthermore, this assay showed 100% positive predictive agreement (PPA) and negative predictive agreement (NPA) relative to SYBR Green qPCR for DTMUV detection in 32 cloacal swabs and 22 tissue samples, supporting its application for clinical field detection.

Highly Sensitive and Quantitative Magnetic Nanoparticle-Based Lateral Flow Immunoassay with an Atomic Magnetometer.

Lateral flow immunoassay (LFIA) is a simple point-of-care method for detecting various analytes. However, the lack of test result precision and poor quantification are the main bottlenecks of LFIA. Although magnetic nanoparticles (MNPs) have gained prominence as potent labels in LIFA, the quantitative detection method for trace biomarkers remains to be improved. Here, we propose a promising real-time biosensing platform based on a highly sensitive atomic magnetometer to fulfill the quantitative detection of MNP-based lateral flow immunochromatographic assays. The strategy entails obtaining the residual flux density component spectrum by continuously and linearly scanning the trace MNP label and then resolving the magnetization and quantity from the spectrum. Moreover, we exploit the theoretical model of the magnetic dipole to verify the method's reliability. Regarding carcinoembryonic antigen detection, the atomic magnetometer exhibits a low detection limit of ∼0.01 ng mL-1 with a 100-fold enhancement factor compared to optical detection methods and a more straightforward mechanism than other magnetic detection approaches. Together, these results provide valuable insight for the potential application of atomic magnetometer quantum measurement techniques in intelligent diagnosis and treatment.

Establishment of a Colloidal Gold Immunochromatographic Method for the Detection of Cypermethrin in Tobacco.

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper.

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Strips for Rapid Detection of Cowpea Mild Mottle Virus.

Cowpea mild mottle virus (CPMMV) is a global plant virus that poses a threat to the production and quality of legume crops. Early and accurate diagnosis is essential for effective managing CPMMV outbreaks. With the advancement in isothermal recombinase polymerase amplification and lateral flow strips technologies, more rapid and sensitive methods have become available for detecting this pathogen. In this study, we have developed a reverse transcription recombinase polymerase amplification combined with lateral flow strips (RT-RPA-LFS) method for the detection of CPMMV, specifically targeting the CPMMV coat protein (CP) gene. The RT-RPA-LFS assay only requires 20 min at 40°C and demonstrates high specificity. Its detection limit was 10 copies/µl, which is approximately up to 100 times more sensitive than RT-PCR on agarose gel electrophoresis. The developed RT-RPA-LFS method offers a rapid, convenient, and sensitive approach for field detection of CPMMV, which contribute to controlling the spread of the virus.

On-site detection of chloramphenicol in fish using SERS-based magnetic aptasensor coupled with a handheld Raman spectrometer.

In recent years, the rapid detection of chloramphenicol (CAP) has become a market demand due to its high toxicity. In this study, for the first time, a portable surface-enhanced Raman scattering (SERS) aptasensor for the rapid and on-site detection of chloramphenicol (CAP) residues in fish was developed. Fe3O4@Au nanoflowers combined with sulfhydryl (SH)-CAP aptamer complementary DNA acted as capture probes. SH-CAP aptamer modified Au@Ag nanoparticles (Au@Ag NPs) embedded with 4-mercaptobenzoic acid (4-MBA) were served as reporter probes. The strongest Raman intensity was produced due to the coupling of Fe3O4@Au nanoflowers (Fe3O4@Au NFs) and Au@Ag NPs. For CAP detection, a wide linear range from 0.001 to 1000 µg/L, with an R2 of 0.9805, was obtained. The limit of detection was determined to be 0.87 ng/L. The SERS aptasensor showed excellent performance for analytical applications for real fish samples. Compared with the conventional HPLC method, the developed SERS aptasensor coupled with a handheld Raman spectrometer had flexible application and avoided the limitations of complex operating conditions. It should be a promising portable analytical tool for analysis of drug residues in the field.

Unbiased Virus Detection in a Danish Zoo Using a Portable Metagenomic Sequencing System.

Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human-animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens (Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time.

Development of a reverse transcription recombinase polymerase amplification combined with lateral flow assay for equipment-free on-site field detection of tomato chlorotic spot virus.

BACKGROUND: Tomato chlorotic spot virus (TCSV) is an economically important, thrips-transmitted, emerging member of the Orthotospovirus genus that causes significant yield loss mainly in tomatoes, but also in other vegetable and ornamental crops. Disease management of this pathogen is often challenging due to the limited availability of natural host resistance genes, the broad host range of TCSV, and the wide distribution of its thrips vector. Point-of-care detection of TCSV with a rapid, equipment-free, portable, sensitive, and species-specific diagnostic technique can provide prompt response outside the laboratory, which is critical for preventing disease progression and further spread of the pathogen. Current diagnostic techniques require either laboratory-dependent or portable electronic equipment and are relatively time-consuming and costly. RESULTS: In this study, we developed a novel technique for reverse-transcription recombinase polymerase amplification combined with lateral flow assay (RT-RPA-LFA) to achieve a faster and equipment-free point-of-care detection of TCSV. The RPA reaction tubes containing crude RNA are incubated in the hand palm to obtain sufficient heat (∼36 °C) for the amplification without the need for equipment. Body-heat mediated RT-RPA-LFA is highly TCSV-specific with a detection limit as low as ∼6 pg/µl of total RNA from TCSV-infected tomato plants. The assay can be performed in 15 min in the field. CONCLUSION: To the best of our knowledge, this is the first equipment-free, body-heat-mediated RT-RPA-LFA technique developed to detect TCSV. Our new system offers a time-saving advantage for the sensitive and specific diagnostic of TCSV that local growers and small nurseries in low-resource settings can use without skilled personnel.

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2-3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions.

Molecular Approaches for Detection of Trichoderma Green Mold Disease in Edible Mushroom Production.

Due to the evident aggressive nature of green mold and the consequently huge economic damage it causes for producers of edible mushrooms, there is an urgent need for prevention and infection control measures, which should be based on the early detection of various Trichoderma spp. as green mold causative agents. The most promising current diagnostic tools are based on molecular methods, although additional optimization for real-time, in-field detection is still required. In the first part of this review, we briefly discuss cultivation-based methods and continue with the secondary metabolite-based methods. Furthermore, we present an overview of the commonly used molecular methods for Trichoderma species/strain detection. Additionally, we also comment on the potential of genomic approaches for green mold detection. In the last part, we discuss fast screening molecular methods for the early detection of Trichoderma infestation with the potential for in-field, point-of-need (PON) application, focusing on isothermal amplification methods. Finally, current challenges and future perspectives in Trichoderma diagnostics are summarized in the conclusions.