Technique For Rapid Detection Research Articles

Field-deployable porcine epidemic diarrhea virus diagnostics utilizing CRISPR-Cas13a

ABSTRACT Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and “collateral” promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT – qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.

Lead acetate-based test strip method for rapid and quantitative detection of residual sulfur dioxide in Chinese herbal medicines.

To prevent the residual sulfur dioxide in Chinese herbal medicines (CHM) caused by sulfur fumigation, which may lead to severe health issues, there is an urgent need for a rapid and quantitative detection technique. Sodium borohydride was used as a reducing agent to convert sulfur dioxide into hydrogen sulfide, which was then detected using lead acetate test strip. An accurate testing apparatus was designed, consisting of reaction bottle cap, reaction bottle, lead acetate test strip, and sulfur dioxide detector. The effect of different reaction variables on detection, including reductant quality, pH of initial media, reaction time, lead acetate concentration, and membrane type was investigated. The optimal conditions were determined by orthogonal experiments. The reaction membrane type and lead acetate concentration on the membrane were optimized to enhance detection accuracy. Standardized gray cards were fabricated and used to calibrate the detector. The detection system demonstrated an exceptional linear correlation (r2 = 0.9992), with a linear detection range of 0-750 mg·kg-1. The colored substances and sulfur-containing substances within the matrix of CHM did not affect the detection results. Therefore, the detection method exhibited superior accuracy and stability. The proposed technique proved to be swift, reliable, and provides a straightforward and convenient approach for the quantitative determination of sulfur dioxide in CHMs. The results of this work may provide insights into the development of test strips for quantitative detection.

Optimization study and luminescence enhancement effect for the determination of moxifloxacin using some rare elements

Moxifloxacin is a potent fluoroquinolone antibiotic and a promising antibacterial drug that significantly reduces skin, pneumonia, and respiratory pathogens. Herein, sensitive fluorescent trivalent lanthanides are desired as a facile, rapid, and effective MOX detection technique using light and heavy rare earth elements, such as Eu3+ and Tb3+, respectively. This spectrofluorometric technique relies on the nonradiative intramolecular energy transfer from MOX to lanthanide ions in the presence of cooperative ligands, for instance, 2,2′-bipyridine, 1,10-phenanthroline, or ethylene diamine, and an anionic surfactant (sodium dodecyl benzene sulfonate SDBS). Optimal fluorescence parameters, for instance, λex, λem, pH, concentrations of MOX, Ln3+, SDBS, and coordinating ligands, were determined based on the extent of sensitization. Micellar-enhanced fluorescence was also studied. The fluorescence intensity of the quaternary MOX-Ln3+-SDBS-phen system dramatically increased with MOX concentrations ranging from 0.4 to 8 µg/mL, with a determination coefficient and detecting limit of 0.9977 and 0.0221 µg/mL, respectively. The emission quantum yields of all systems were calculated, and it was observed that MOX-Tb3+-phen-SDBS has the highest quantum yield (0.26). Furthermore, the performance of this precise technique for detecting MOX was confirmed in pharmaceutical tablets and human serum/urine samples. The satisfactory recoveries% and RSD were found from 99.25 to 100.5 % and 1.6 to 1.8 % in tablets and from 95.33 to 103.35 % with 0.7 to 2.9 % in human samples, respectively.

Surface-enhanced Raman spectroscopy for the rapid identification of fosfomycin resistant and sensitive strains of E. Coli

Development of rapid detection and discrimination technique for the antibiotic resistant and sensitive bacterial strains is required for this purpose, Surface-enhanced Raman spectroscopy (SERS) technique is considered to have great potential. To develop a fast and sensitive detection and discrimination methodology based on SERS technique along with chemometric tools for the differentiation among fosfomycin sensitive and resistant Escherichia coli (E. coli.) strains. In this research work, three E. Coli strains resistant to fosfomycin with three fosfomycin sensitive E. coli strains were characterized in which silver nanoparticles were employed as SERS substrate. Moreover, MATLAB 7.8 (2009a) was used for the preprocessing, baseline correction and normalization of SERS spectra. Using principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA), several E. Coli strains were identified on the basis of their distinctive SERS spectral properties. The differentiating SERS spectral features which can be associated with the resistance and sensitivity against fosfomycin antibiotic, are obtained by comparing mean SERS spectrum of strains of resistant E. coli with sensitive E. coli strains. Chemometric techniques, such as principal components analysis, have been proven effective for qualitative analysis. Additionally, PLSDA was employed to classify the SERS spectra acquired from the pellets of several bacterial strains. SERS is a useful analytical method for quickly differentiating between E. Coli strains that are sensitive to fosfomycin and those that are resistant to it.

Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 104 copies/μL and 103 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2–3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.

Combining NIR spectroscopy with chemometrics for discriminating naturally ripened banana and calcium carbide ripened banana

Calcium carbide is prohibited as a fruit ripening agent in many countries due to its harmful effects. Current methods for detecting calcium carbide in fruit involve time-consuming and destructive chemical analysis techniques, necessitating the need for non-destructive and rapid detection techniques. This study combined near infrared (NIR) spectroscopy with chemometrics to detect two banana varieties ripened with calcium carbide in different forms when they are peeled or unpeeled. Sixteen linear discriminant analysis (LDA) models were developed with high average classification accuracies for classifying banana based on the mode used to ripen banana, type of carbide treatment and the duration of soaking banana in carbide solution. Banana colour was predicted with partial least squared regression (PLSR) models with R2CV > 0.74, RMSECV and <5.4 and RPD close to 3. NIR coupled with chemometrics has good potential as a technique for detecting carbide ripened banana even if the banana is peeled or not.

Estimation of the soluble solid content of citrus based on the fractional-order derivative and optimal band combination algorithm.

The soluble solid content (SSC) is a primary characteristic index for evaluating the internal quality of citrus fruits. The development of rapid and nondestructive SSC detection techniques can help address the current issues of postharvest quality grading in China's citrus industry. In this study, a total of 261 experimental samples, including 70 Murcott, 91 Clementine, and 100 Navel orange, were divided into prediction and validation sets in a 7:3 ratio. After obtaining the reflection spectra and SSCs, SNV-FOD (Standard Normal Variate-Fractional-Order Derivative) was used to process the spectra, and the optimal band combination algorithm was introduced to select SSC-sensitive bands. Then, the obtained optimal dual-band combination was input into eight regression models for comparison, and the best performing models stacked ensemble models was selected. Finally, the H-ELR (HyperOpt-optimized ensemble learning regression) model, optimized using a Bayesian function, was applied for the effective estimation of SSC for three common citrus varieties in Guangxi, Murcott, Clementine, and Navel oranges. The results show that (1) the SNV-FOD preprocessing method proposed in this study improved the correlation coefficient with the SSC from 0.546 to 0.836 compared to that of the original spectrum, (2) the optimal dual-band combination (969 and 1069nm) constructed by integrating the differential index and 1.2-order derivative yielded the most accurate results (RPD=2.13), and (3) the H-ELR model, based on HyperOpt optimization, achieved good estimated performance (RPD=2.46). PRACTICAL APPLICATION: This research contributes to the development of practical SSC prediction instruments with excellent universality and ease of application.

Detection of Mycotoxin Contamination in Foods Using Artificial Intelligence: A Review.

Mycotoxin contamination of foods is a major concern for food safety and public health worldwide. The contamination of agricultural commodities employed by humankind with mycotoxins (toxic secondary metabolites of fungi) is a major risk to the health of the human population. Common methods for mycotoxin detection include chromatographic separation, often combined with mass spectrometry (accurate but time-consuming to prepare the sample and requiring skilled technicians). Artificial intelligence (AI) has been introduced as a new technique for mycotoxin detection in food, providing high credibility and accuracy. This review article provides an overview of recent studies on the use of AI methods for the discovery of mycotoxins in food. The new approach demonstrated that a variety of AI technologies could be correlated. Deep learning models, machine learning algorithms, and neural networks were implemented to analyze elaborate datasets from different analytical platforms. In addition, this review focuses on the advancement of AI to work concomitantly with smart sensing technologies or other non-conventional techniques such as spectroscopy, biosensors, and imaging techniques for rapid and less damaging mycotoxin detection. We question the requirement for large and diverse datasets to train AI models, discuss the standardization of analytical methodologies, and discuss avenues for regulatory approval of AI-based approaches, among other top-of-mind issues in this domain. In addition, this research provides some interesting use cases and real commercial applications where AI has been able to outperform other traditional methods in terms of sensitivity, specificity, and time required. This review aims to provide insights for future directions in AI-enabled mycotoxin detection by incorporating the latest research results and stressing the necessity of multidisciplinary collaboration among food scientists, engineers, and computer scientists. Ultimately, the use of AI could revolutionize systems monitoring mycotoxins, improving food safety and safeguarding global public health.

Strip biosensors based on broad-spectrum aptamers and cationic polymers for the on-site rapid detection of tetracycline antibiotics residues in milk

A lateral flow chromatography strip (LFS) is a chromatography-based biosensor with advantages of convenient portability, simple operation and rapid detection. In this study, a novel rapid detection technique of aptamer-based chromatography strip was developed and used for the first time for the residue detection of tetracycline antibiotics (TCs) in various milk samples. In this method, gold nanoparticles (AuNPs) modified by TCs specific aptamers were used as probes, and cationic polymers as capture molecules in a test line (T-line). Meanwhile, the analysis of the gray value of the T-line enabled a linear detection range of 1–300 nM and a limit of detection (LOD) of 0.33 nM for quantitative analysis. The biosensor demonstrated high specificity, good stability, and successfully detected tetracyclines in milk samples with recovery rate of 93.60%–106.20%. This method sets a basis for multi-residue antibiotics detection in diverse samples, showing potential for on-site applications.

Rapid and Sensitive On-site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.

Maize stalk rot is a soil-borne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, Fusarium proliferatum, and Fusarium graminearum. Based on TEF-1α, we developed a rapid detection technique using RPA-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/μL, 0.11 ng/μL, and 0.13 ng/μL, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.

GeneXpert Assay in Detection of Pulmonary Tuberculosis and Rifampicin Tuberculosis Resistant in Tehsil Hazro Population; A Cross Sectional Study

Objective: To determine the prevalence of Tuberculosis and MDR Tuberculosis by GeneXpert assay in Tehsil Hazro District Attock. Study design: cross sectional study Place and duration of study: Study was carried out at THQ hospital Hazro of District Attock from January 2019 to December 2020 Patients and Methods: Total 1460 patients of TB suspect were included in the study. First LED microscopy of all TB suspects was done. LED Microscopy positive cases along with suspects having history of HIV, close contacts of MDR TB, all TB cases of retreatment and sputum-negative cases of LED microscopy having positive clinical findings were sent for GeneXpert Assay. Results from the GeneXpert assay were categorized as GeneXpert positive, GeneXpert negative, and MDR Tuberculosis. Determinants of MDR- TB were identified using questionnaires regarding MDR –TB. Results: Out of total 205 patients on GeneXpert Assay, 43 patients were GeneXpert positive test with no rifampicin resistance, 3 patients were GeneXpert positive with rifampicin resistance and 63 patients were GeneXpert negative but sign and symptoms, X-ray findings and laboratory tests were supporting tuberculosis and according to opinion of TB specialist they were labeled as Sputum negative Tuberculosis. Tuberculosis prevalence in Tehsil Hazro was 0.073. From the results, it was concluded that an overall MDR rate was 2.7% in our study population. Conclusion: GeneXpert Assay is a rapid technique and reliable for Mycobacterium detection. The main determinants of MDR-TB were the patients who had contact h/o with MDR cases and those who did not follow the treatment regime in their previous Tuberculosis treatment. Keywords: GeneXpert Assay, LED microscopy, MDR Tuberculosis, Sputum positive TB, Sputum negative TB.

Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium.

One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

Rapid detection and occurrence of foodborne pathogens in minimally processed vegetables: a review

SummaryThe increased consumption of minimally processed vegetables (MPV) in various countries is related to the continued interest of consumers in seeking practical and healthy food items. Due to multiple processing steps, MPV can be contaminated by several foodborne pathogens that pose significant health risks to consumers. The use of rapid techniques to detect pathogens in ready‐to‐eat (RTE) foods such as MPV is therefore essential to provide high quality and safe products. This review aims to provide a comprehensive description of molecular‐based techniques for rapid detection of pathogenic bacteria in MPV, and their occurrence reported in studies published in the last 10 years. The main pathogens detected using rapid methods were Salmonella spp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Shigella spp., and Campylobacter jejuni. Molecular‐based techniques included real‐time polymerase chain reaction (PCR), multiplex PCR, matrix‐assisted laser desorption ionisation time of flight mass spectrometry (MALDI‐TOF MS), and denaturing gradient gel electrophoresis (DGGE). The data indicate high incidences of pathogenic bacteria in MPV, stressing the need for their rapid detection in these products to prevent associated health risks. Further studies should be carried out to increase the sensitivity of molecular‐based techniques and prevent false positives due to undesirable non‐specific PCR amplifications.

Correction methods for visible near-infrared spectroscopy in water pH detection to mitigate temperature influence

The pH value is an important indicator for determining water quality, and visible near-infrared (Vis-NIR) spectroscopy is an ideal technique for rapid detection. The Vis-NIR spectrum of water is greatly affected by temperature. When using Vis-NIR spectroscopy to detect the pH value of water, temperature interference will affect the prediction accuracy of the water pH value. In this study, several chemometric methods developed in recent years were used to correct the spectra of distilled water samples under temperature interference to improve the prediction accuracy of water pH values. Specifically, this includes the external parameter orthogonalization (EPO) algorithm and the generalized least square weighting (GLSW) algorithm. Spectra of distilled water samples at different temperatures in the 400–1049 nm range were collected, and the correction effects of the EPO algorithm and the GLSW algorithm on the spectra after temperature changes, as well as the prediction errors of water pH values, were tested and examined, compared with traditional full-temperature spectral correction models. The results show that both the EPO algorithm and the GLSW algorithm methods require only a small number of representative samples to obtain filtering matrices. By correcting the spectra of distilled water samples at all temperatures to the reference temperature level, good correction effects can be achieved. Additionally, the prediction accuracy of water pH values of the models corrected by both methods is better than that of the traditional method, and the complexity of the models is also greatly reduced.

Pursuing drug laboratories: Analysis of drug precursors with High Kinetic Energy Ion Mobility Spectrometry

High Kinetic Energy Ion Mobility Spectrometry (HiKE-IMS) is a technique for rapid and reliable detection of trace compounds down to ppbV-levels within one second. Compared to classical IMS operating at ambient pressure and providing the ion mobility at low electric fields, HiKE-IMS can also provide the analyte-specific field dependence of the ion mobility and a fragmentation pattern at high reduced electric field strengths. The additional information about the analyte obtained by varying the reduced electric field strength can contribute to reliable detection. Furthermore, the reduced number of ion-molecule reactions at the low operating pressure of 10 – 40 mbar and the shorter reaction times reduce the impact of competing ion-molecule reactions that can cause false negatives. In this work, we employ HiKE-IMS for the analysis of phenyl-2-propanone (P2P) and other precursor chemicals used for synthesis of methamphetamine and amphetamine. The results show that the precursor chemicals exhibit different behavior in HiKE-IMS. Some precursors form a single significant ion species, while others readily form a fragmentation pattern. Nevertheless, all drug precursors can be distinguished from each other, from the reactant ions and from interfering compounds. In particular, the field-dependent ion mobility as an additional separation dimension aids identification, potentially reducing the number of false positive alarms in field applications. Furthermore, the analysis of a seized illicit P2P sample shows that even low levels of P2P can be detected despite the complex background present in the headspace of real samples.

Characterization, Molecular Mechanism of Prochloraz-Resistance in Fusarium fujikuroi and Development of Loop-Mediated Isothermal Amplification Rapid Detection Technique Based on the S312T Genotype of Resistances.

Rice bakanae disease (RBD) is a typical seed-borne fungal disease caused by Fusarium fujikuroi. Prochloraz is a sterol demethylation inhibitor, which is among the most important classes of active ingredients for the management of RBD. In 2022, the total resistance frequency of F. fujikuroi to prochloraz in Zhejiang Province was 62.67%. The fitness of the prochloraz-resistant population was lower than that of the susceptible population, but its pathogenicity was slightly stronger. The S312T and F511S double mutations of Ffcyp51b were detected in the resistant isolates. Loop-mediated isothermal amplification (LAMP) technology based on S312T was established to rapidly determine prochloraz resistance in F. fujikuroi. LAMP primer mismatch design was performed based on the cyp51b gene, and 100-300 bp sequences containing a mutation at codon 312 were amplified. In a 25 µL reaction tube, 1 pg/µL DNA of F. fujikuroi could be detected. The detection limit for the frequency of prochloraz resistance was 0.498% using this method. We performed LAMP detection on rice seedlings inoculated with prochloraz-sensitive and -resistant isolates and treated them with prochloraz. Prochloraz demonstrated good control in rice seedlings. A chromogenic reaction was observed in seedlings treated with prochloraz-resistant isolates, and the results were verified using electrophoresis. It has been demonstrated that LAMP technology based on the S312T genotype can quickly and specifically detect prochloraz-resistant isolates in rice seedlings.

A hyperspectral imaging technique for rapid non-destructive detection of soluble solid content and firmness of wolfberry

A hyperspectral imaging technique for rapid non-destructive detection of soluble solid content and firmness of wolfberry

Validation of a rapid collagenase activity detection technique based on fluorescent quenched gelatin with synovial fluid samples

BackgroundMeasuring collagenase activity is crucial in the field of joint health and disease management. Collagenases, enzymes responsible for collagen degradation, play a vital role in maintaining the balance between collagen synthesis and breakdown in joints. Dysregulation of collagenase activity leads to joint tissue degradation and diseases such as rheumatoid arthritis and osteoarthritis. The development of methods to measure collagenase activity is essential for diagnosis, disease severity assessment, treatment monitoring, and identification of therapeutic targets.ResultsThis study aimed to validate a rapid collagenase activity detection technique using synovial fluid samples. Antibody microarray analysis was initially performed to quantify the levels of matrix metalloproteinase-9 (MMP-9), a major collagenase in joints. Subsequently, the developed gelatin-based test utilizing fluorescence measurement was used to determine collagenase activity. There was a significant correlation between the presence of MMP-9 and collagenase activity. In addition, Lower Limit of Detection and Upper Limit of Detection can be preliminary estimated as 8 ng/mL and 48 ng/mL respectively.ConclusionsThe developed technique offers a potential point-of-care assessment of collagenase activity, providing real-time information for clinicians and researchers. By accurately quantifying collagenase activity, healthcare professionals can optimize patient care, improve treatment outcomes, and contribute to the understanding and management of joint-related disorders. Further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.

A Glycoprotein-Based Surface-Enhanced Raman Spectroscopy-Lateral Flow Assay Method for Abrin and Ricin Detection.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick

Tick-borne encephalitis virus (TBEV), a zoonotic pathogen, can cause severe neurological complications and fatal outcomes in humans. Early diagnosis of TBEV infection is crucial for clinical practice. Although serological assays are frequently employed for detection, the lack of antibodies in the early stages of infection and the cross-reactivity of antibodies limit their efficacy. Conventional molecular diagnostic methods such as RT-qPCR can achieve early and accurate identification but require specialized instrumentation and professionals, hindering their application in resource-limited areas. Our study developed a rapid and visual TBEV molecular detection method by combining RT-recombinase-aided amplification, the CRISPR/Cas13a system, and lateral flow dipsticks. The diagnostic sensitivity of this method is 50 CFU/ml, with no cross-reactivity with a variety of viruses. The detection can be carried out within 1 h at a temperature between 37 and 42 °C, and the results can be visually determined without the need for complex instruments and professionals. Subsequently, this assay was used to analyze clinical samples from 15 patients suspected of TBEV infection and 10 healthy volunteers, and its sensitivity and specificity reached 100 %, which was consistent with the results of RT-qPCR. These results indicate that this new method can be a promising point-of-care test for the diagnosis of tick-borne encephalitis.