Lanthanide metal-organic frameworks as ratiometric fluorescent probes for real-time monitoring of PFOA photocatalytic degradation process.

The assessment of perfluorooctanoic acid (PFOA) photocatalytic degradation usually involves tedious pre-treatment and sophisticated instrumentation, making it impractical to evaluate the degradation process in real-time. Herein, we synthesized a series of lanthanide metal-organic frameworks (Ln-MOFs) with outstanding fluorescent sensing properties and applied them as luminescent probes in the photocatalytic degradation reaction of PFOA for real-time evaluation. As the catalytic reaction proceeds, the fluorescence color changes significantly from green to orange-red due to the different interaction mechanisms between the electron-deficient PFOA and smaller radius F- with the ratiometric fluorescent probe MOF-76 (Tb: Eu = 29:1). The limit of detection (LOD) was calculated to be 0.0127 mM for PFOA and 0.00746 mM for F-. In addition, the conversion rate of the catalytic reaction can be read directly based on the chromaticity value by establishing a three-dimensional relationship graph of G/R value-conversion rate-time (G/R indicates the ratio between green and red luminance values in the image.), allowing for real-time and rapid tracking of the PFOA degradation. The recoveries of PFOA and F- in the actual water samples were 99.3-102.7% (RSD = 2.2-4.4%) and 100.7-105.3% (RSD = 3.9-6.8%), respectively. Both theoretical calculations and experiments reveal that the detection mechanism was attributed to the photoinduced electron transfer and energy transfer between the analytes and the probe. This method simplifies the sample analysis process and avoids the use of bulky instruments, and thus has great potential on the design and development of quantitative time-resolved visualization methods to assess catalytic performance and reveal mechanisms.

Improving the Efficiency of Luminescent Zn(II)-Modified N-Doped GOQD Nanomaterials in Parkinson's Disease Treatment: A Theoretical Mechanistic Framework Exploring Doping Effect.

Levodopa, a widely prescribed drug in Parkinson's disease treatment, stands as the foremost prodrug of dopamine. An affordable self-testing kit is utilized to monitor  levodopa content in anti-parkinson drugs in human serum. A photoluminescent trinuclear Zn(II) complex [Zn3(L)2(κ1-OAc)2(κ2-OAc)2] has been synthesized, which cleaves into mononuclear  ZC in aqueous solution. ZC was found to detect L-Dopa in Tris-HCl buffer, exhibiting a moderate decrease in PL-emission. The real-life utility of the ZC probe is limited, for its lower sensitivity (LOD 35.3 µM) and separation challenges. Therefore, an interface between homogeneous and heterogeneous supports has been explored, leading to the strategic development of NGOZC, where ZC was grafted onto NGOQD. This material enables naked- eye detection under both ambient and UV light with color change from bright cyan to green, followed by dark. The nitrogen doping effect was investigated by several comparative investigations involving the synthesis of ZC-grafted GOQD, leading to enhanced quenching performance. Steady-state and time-resolved fluorescence titration study, morphological analysis, and computational calculations have been performed to get insights into the sensing mechanism. To the best of our knowledge, this as-synthesized NGOZC (LOD 1.78 nM) represents a promising strategy and platform for applications in biosensors, especially for Parkinson's and Alzheimer's diseases.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification.

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/µL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/µL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

Neural Network Enables High Accuracy for Hepatitis B Surface Antigen Detection with a Plasmonic Platform.

The detection of hepatitis B surface antigen (HBsAg) is critical in diagnosing hepatitis B virus (HBV) infection. However, existing clinical detection technologies inevitably cause certain inaccuracies, leading to delayed or unwarranted treatment. Here, we introduce a label-free plasmonic biosensing method based on the thickness-sensitive plasmonic coupling, combined with supervised deep learning (DL) using neural networks. The strategy of utilizing neural networks to process output data can reduce the limit of detection (LOD) of the sensor and significantly improve the accuracy (from 93.1%-97.4% to 99%-99.6%). Compared with widely used emerging clinical technologies, our platform achieves accurate decisions with higher sensitivity in a short assay time (∼30 min). The integration of DL models considerably simplifies the readout procedure, resulting in a substantial decrease in processing time. Our findings offer a promising avenue for developing high-precision molecular detection tools for point-of-care (POC) applications.

A Fluorescent Organic Probe for Naked-Eye Detection of Chloromethanes.

An organic fluorescent probe (OFP-TAR) with a propeller-like structure was designed and synthesized. The photoluminescence of OFP-TAR in solution exhibited a significant red shift with the increase of solvent polarity, enabling a transition of fluorescence emission from blue (445 nm) to yellow (540 nm). The organic thin-film materials based on OFP-TAR/PMMA exhibit significant color changes upon exposure to CH2Cl2, CHCl3, and CCl4, with their maximum fluorescence wavelengths measured at 445, 471, and 494 nm respectively. The device facilitates the visual detection of chloromethanes and is capable of enduring more than 7 cycles of testing. These materials can also be prepared as binary-coded microarray data storage devices or applied in the field of anti-counterfeiting. The quantum yields of guest-loaded crystals CH2Cl2@OFP-TAR, CHCl3@OFP-TAR and CCl4@OFP-TAR are observed as 19.13 %, 8.79 %, and 0.83 % respectively, which are consistent with the tendency of OFP-TAR in CH2Cl2 (47.30 %), CHCl3 (34.27 %) and CCl4 (3.10 %). The fluorescence properties of OFP-TAR, OFP-TAR/PMMA, guest-loaded and guest-free crystals provided insights into the special response mechanism of OFP-TAR towards different chloromethanes.

Surface-Imprinted Polysiloxane with Recognition Ability Based on an ITO Layer for Rapid Detection of Fusarium oxysporum f. sp. cubense by the Naked Eye.

Direct observation by the naked eye of fluorescence-stained microbes adsorbed on surface imprinted polymers (SIPs) is highly challenging and limited by speed, accuracy and the semiquantitative nature of the method. In this study, we tested for the presence of spores of Fusarium oxysporum f. sp. cubense race 4 (Foc4), which cause severe banana Fusarium wilt disease and reduces the area of banana plants. This kind of spore can become dormant in soil, which means that the detection of secreted molecules (molecular imprinting) in soil may be inaccurate; detection methods such as polymerase chain reaction (PCR) and Raman spectroscopy are more accurate but time-consuming and inconvenient. Therefore, a semiquantitative and rapid SIP detection method for Foc4 was proposed. Based on the ITO conductive layer, a reusable and naked-eye-detectable Foc4-PDMS SIP film was prepared with a site density of approximately 9000 mm-2. Adsorption experiments showed that when the Foc4 spore concentration was between 104 to 107 CFU/mL, the number of Foc4 spores adsorbed and the fluorescence intensity were strongly correlated with the concentration and could be fully distinguished by the naked eye after fluorescence staining. Adsorption tests on other microbes showed that the SIP film completely recognized only the Foc series. All the results were highly consistent with the naked-eye observations after fluorescence staining, and the results of the Foc4-infected soil experiment were also close to the ideal situation. Taken together, these results showed that Foc4-PDMS SIPs have the ability to rapidly and semiquantitatively detect the concentration of Foc in soil, which can provide good support for banana cultivation. This method also has potential applications in the detection of other fungal diseases.

A Zn-MOF-based mixed matrix membrane as an ultrastable luminescent sensor for selective and visual detection of antibiotics and pesticides in food samples.

The detection of antibiotics and pesticides are of great significance since their residues threaten the health of human beings by accumulation. However, most traditional solid chemical sensors are suffer from the limitations of low sensitivity and economic practicability because of the aggregating nature and unstable of solid sensors. Herein, a new luminescent sensor 1@PMMA (1, [(ZnL)·H2O]n (H2L = 5-(4-(pyridin-4-yl)benzamido)benzene-1,3-dioic acid); PMMA = poly(methyl methacrylate)) was successfully prepared. Notably, the polymer matrix provided the chemical protection for MOF particles. The as fabricated 1@PMMA was stable in milk, honey and egg as well as exhibited strong blue emission under ultraviolet light irradiation, which can act as luminescent probe for detecting antibiotics and pesticides. More interestingly, 1@PMMA exhibited visual, real-time and recyclable detection of antibiotics ornidazole (ODZ) and pesticides 2,6-dichloro-4-nitrobenzenamine (DCN) in real food samples. This work shows that the luminescent MOF-based mixed matrix membranes could be applied as good candidates for sensing analytes in practical application.

Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.

Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10-2 ng µL-1 and 102 CFU mL-1, respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

Solvent-tuned perovskite heterostructures enable visual linoleic acid assay and edible oil species discrimination via wavelength shift.

Linoleic acid (LA) detection and edible oils discrimination are essential for food safety. Recently, CsPbBr3@SiO2 heterostructures have been widely applied in edible oil assays, while deep insights into solvent effects on their structure and performance are often overlooked. Based on the suitable polarity and viscosity of cyclohexane, we prepared CsPbBr3@SiO2 Janus nanoparticles (JNPs) with high stability in edible oil and fast halogen-exchange (FHE) efficiency with oleylammonium iodide (OLAI). LA is selectively oxidized by lipoxidase to yield hydroxylated derivative (oxLA) capable of reacting with OLAI, thereby bridging LA content to naked-eye fluorescence color changes through the anti-FHE reaction. The established method for LA in edible oils exhibited consistent results with GC-MS analysis (p > 0.05). Since the LA content difference between edible oils, we further utilized chemometrics to accurately distinguish (100%) the species of edible oils. Overall, such elaborated CsPbBr3@SiO2 JNPs enable a refreshing strategy for edible oil discrimination.

Linker-Preserved Iron Metal-Organic Framework-Based Lateral Flow Assay for Sensitive Transglutaminase 2 Detection in Urine Through Machine Learning-Assisted Colorimetric Analysis.

A groundbreaking demonstration of the utilization of the metal-organic framework MIL-101(Fe) as an exceptionally perceptive visual label in colorimetric lateral flow assays (LFA) is described. This pioneering approach enables the precise identification of transglutaminase 2 (TGM2), a recognized biomarker for chronic kidney disease (CKD), in urine specimens, which offers a remarkably sensitive naked-eye detection mechanism. The surface of MIL-101(Fe) was modified with oxalyl chloride, adipoyl chloride, and poly(acrylic) acid (PAA); these not only improved the labeling material stability in a complex matrix but also achieved a systematic control in the detection limit of the TGM2 concentration using our LFA platform. The advanced LFA with the MIL-101(Fe)-PAA label can detect TGM2 concentrations down to 0.012, 0.009, and 0.010 nM in Tris-HCl buffer, urine, and desalted urine, respectively, which are approximately 55-fold lower than those for a conventional AuNP-based LFAs. Aside from rapid TGM2 detection (i.e., within 20 min), the performance of the MIL-101(Fe)-PAA-based LFA on reproducibility [coefficients of variation (CV) < 2.9%] and recovery (95.9-103.2%) along with storage stability within 25 days of observation (CV < 6.0%) shows an acceptable parameter range for quantitative analysis. A sophisticated sensing method grounded in machine learning principles was also developed, specifically aimed at precisely deducing the TGM2 concentration by analyzing immunoreaction sites. More importantly, our developed LFA offers potential for clinical measurement of TGM2 concentration in normal human urine and CKD patients' samples.

Naked-eye sensitive detection of nanoPET by pH-responsive colorimetric method based on dual-enzyme catalysis.

A pH-responsive colorimetric method based on dual-enzyme catalysis for rapid and facile detection and quantification of nanoPET at environment-dependent concentration is proposed. The nanoPET was hydrolyzed by the synergistic catalysis of cutinase and lipase to terephthalic acid which can be sensitive detected using bromocresol purple as the indicator. The color changed from purple to bright yellow as the nanoPET detection concentration increased from 0 mg/mL to 2 mg/mL which can be detected by UV-Vis. This naked-eye method has a high sensitivity for nanoPET detection with the visual detection cutoff of 31.00 µg/mL, and has a good linearity in the range of 0 âˆ¼ 1 mg/mL with LOD of 22.84 µg/mL. The reliability of this method is verified in the detection of nanoPET in lake water and beer samples, with an average recovery of 87.1 %. The as-developed dual-enzyme colorimetric chemosensor holds promising potential as a robust and effective platform for the sensitive detection of nanoPET.

Eggshell membrane as a novel and green platform for the preparation of highly efficient and reversible curcumin-based colorimetric sensor for the monitoring of chicken freshness.

Herein, for the very first time, we report a paper-like biomass, eggshell membrane (ESM), as a suitable platform for the fabrication of a colorimetric sensor (E-Cot). Green ethanolic extract, curcumin (CUR), was used as a sensing material to coat with the ESM. The present E-Cot effectively changed its color (yellow to red) in the real-time monitoring for chicken spoilage. The E-Cot exhibits barrier properties due to its inherent semi-permeability characteristics. Interestingly, the E-Cot showed a significant change in total color difference value (ΔE, 0 days - 0.0-39.6, after 1 day - 39.6-42.1, after 2 days - 42.1-53.6, after 3 days- 53.6-60.1, and after 4 days - 60.1-66.3, detectable by the naked eye) in the real-time monitoring for chicken freshness. In addition, the present E-Cot smart colorimetric sensor is reversible with a change in pH, and the sensor can be reused. Further, the hydrophobic nature of the E-Cot was confirmed by water contact angle analysis (WCA, contact angle of 101.21 ± 8.39). Good antibacterial, barrier, and optical properties of the present E-Cot were also found. Owing to the advantages such as green, efficient, cost-effective, biodegradable, reusable, sustainable, and simple preparation, we believe that the present E-Cot would be a more attractive candidate.

A Chromogenic Probe for Detection of Biologically Important Anions and its Implications for Designing Molecular Logic Gates.

Detection of fluoride (F-), acetate (AcO-), and cyanide (CN-) anions is vital from the biological and environmental aspects. In the present contributions, we have introduced a simple Salen-type chromogenic sensor, BEN, to detect these biologically important anions. Changes in UV-visible absorption spectra and color of BEN solution from very pale yellow to pink color are similar for each of these anions and found to be reversible only in the case of F- ions in attendance of HSO4- ions. The estimated limit of detection of BEN solution for detecting F-, AcO-, and CN- anions is found to be below the micromolar (µM) concentration level. Our fabricated handy paper test kit is suitable for qualitatively naked-eye detection of the anions. An immediate quantitative estimation of these important anions is possible using our BEN employing a smartphone, avoiding any costly experimental setup.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens.

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

A "Pincer" Type of Acridine-Triazole Fluorescent Dye for Iodine Detection by Both 'Naked-Eye' Colorimetric and Fluorometric Modes.

Iodine, primarily in the form of iodide (I-), is the bioavailable form for the thyroid in the human body. Both deficiency and excess intake of iodide can lead to serious health issues, such as thyroid disease. Selecting iodide ions among anions has been a significant challenge for decades due to interference from other anions. In this study, we designed and synthesized a new pincer-type acridine-triazole fluorescent probe (probe 1) with an acridine ring as a spacer and a triazole as a linking arm attached to two naphthol groups. This probe can selectively recognize iodide ions in a mixed solvent of THF/H2O (v/v, 9/1), changing its color from colorless to light yellow, making it suitable for highly sensitive and selective colorimetric and fluorescent detection in water systems. We also synthesized another molecular tweezer-type acridine-triazole fluorescent probe (probe 2) that exhibits uniform detection characteristics for iodide ions in the acetonitrile system. Interestingly, compared to probe 2, probe 1 can be detected by the naked eye due to its circulation effect, providing a simple method for iodine detection. The detection limit of probe 1 is determined to be 10-8 mol·L-1 by spectrometric titration and isothermal titration calorimetry measurements. The binding stoichiometry between probe 1 and iodide ions is calculated to be 1:1 by these methods, and the binding constant is 2 × 105 mol·L-1.

Synthesis of photo- and ionochromic N-acylated 2-(aminomethylene)benzo[b]thiophene-3(2Н)-ones with a terminal phenanthroline group.

A series of novel photo- and ionochromic N-acylated 2-(aminomethylene)benzo[b]thiophene-3(2Н)-ones with a terminal phenanthroline receptor substituent was synthesized. Upon irradiation in acetonitrile or DMSO with light of 436 nm, they underwent Z-E isomerization of the C=C bond, followed by very fast N→O migration of the acyl group and the formation of nonemissive O-acylated isomers. These isomers were isolated preparatively and fully characterized by IR, 1H, and 13C NMR spectroscopy as well as HRMS and XRD methods. The reverse thermal reaction was catalyzed by protonic acids. N-Acylated compounds exclusively with Fe2+ formed nonfluorescent complexes with a contrast naked-eye effect: a color change of the solutions from yellow to dark orange. Subsequent selective interaction with AcO- led to the restoration of the initial absorption and emission properties. Thus, the obtained compounds represent dual-mode "on-off-on" switches of optical and fluorescent properties under sequential exposure to light and H+ or sequential addition of Fe2+ and AcO- ions.

[Applications of Naked-Eye 3D Display Technology in Medical Field].

Naked-eye 3D display technology has excellent 3D visual effects and does not require wearable devices assistance. It can present the depth, position and complex structure information of 3D medical images, allowing viewers to obtain information about tissues and organs from different points, reducing cognitive load, contributing to medical teaching and opening up innovative methods for planning and diagnosis. Naked-eye 3D augmented reality display can display medical images in real 3D space, achieving virtual and real vision. It helps a lot to medical research. The applications of naked-eye 3D display technology in three major aspects of medical diagnosis, clinical surgery and rehabilitation training is reviewed in the study. It provides the direction for the subsequent research in medical field, thus assisting medical research and improving medical practice.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.