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Mycoplasma hyopneumoniae (M. hyopneumoniae) is a significant porcine respiratory disease complex pathogen, prompting many swine farms and production systems to pursue M. hyopneumoniae elimination strategies. Antibody testing is cost-effective in demonstrating sustained freedom from M. hyopneumoniae, often replacing PCR testing on deep tracheal swabs. The process typically involves testing a subpopulation of the herd using an M. hyopneumoniae screening antibody ELISA, with non-negative results further assessed through confirmatory testing, such as PCR. Recently, a commercial (Biochek) fluorescent microsphere immunoassay (FMIA) for detecting M. hyopneumoniae antibodies has been introduced as an alternative to ELISA. Its performance was compared to three commercial ELISAs (Idexx, Hipra, and Biochek) using experimental serum samples from pigs inoculated with M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. flocculare, or mock-inoculated with Friis medium. FMIA consistently detected M. hyopneumoniae at earlier time points than the ELISAs, although two false-positive results were encountered using the manufacturer's recommended cutoff. ROC analysis allowed for the evaluation of various cutoffs depending on testing objectives. Poisson regression of misclassification error counts detected no difference in the Biovet FMIA and Hipra ELISA but significantly fewer misclassification errors than Idexx and Biocheck ELISAs. This study showed FMIA as a suitable alternative to traditional ELISAs for screening purposes due to its superior antibody detection rate at early stages. Alternatively, adopting a more stringent cutoff to improve diagnostic specificity could position the FMIA as a viable confirmatory test option. Overall, FMIA is an optimal choice for M. hyopneumoniae antibody surveillance testing, offering versatility in testing strategies (e.g., triplex FMIA M. hyopneumoniae/PRRSV types 1 and 2) and contributing to improved diagnostic capabilities in porcine health management.

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Time-resolved fluorescent lateral immunoassay strip (TRFLIS) is a reliable and rapid method for detecting acetamiprid. However, its sensitivity is often affected by the structural patterns and stability of the fluorescent probe. Researchers have shown significant interests in using goat anti-mouse IgG (GaMIgG) which is indirectly bound to time-resolved fluorescent microsphere (TRFM) and antibody. This allowed for oriented modification of the antibody. However, the stability of fluorescent probe in this binding mode remained unexplored. Herein, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was innovatively used as a cross-linking agent to enhance the binding of antibody to GaMIgG, which improved the stability of the fluorescent probe. Under optimal working conditions, this strategy exhibited a wide linear response range of 5-700 ng/mL. Its limit of detection (LOD) was 0.62 ng/mL, the visual LOD was 5 ng/mL, and the limit of quantification (LOQ) of 2.06 ng/mL. Additionally, under tomato matrix, leek matrix and Chinese cabbage matrix, the linear response ranges were 5-400, 5-300, and 5-700 ng/mL, with LODs of 0.16, 0.60, and 0.41 ng/mL, with LOQs of 0.53, 2.01 and 1.37 ng/mL, respectively. In conclusion, this strategy effectively reduced the dosage of acetamiprid antibody compared with TRFM directly linking acetamiprid antibody, and greatly increased the sensitivity of TRFLIS. Meanwhile, it demonstrated outstanding specificity and accuracy in acetamiprid detection and had been successfully applied to vegetable samples. This method enables rapid and accurate detection of large-volume samples by combining qualitative and quantitative methods. As such, it has great potential in the development of low-cost and high-performance immunochromatographic platforms.

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Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

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A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O.tsutsugamushi were sprayed in another line across the membrane making this the "Control" line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the "Test" line if the sample contains antibodies to O.tsutsugamushi. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus.

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Objective To develop a time-resolved fluorescent immunoassay kit for the rapid,accurate and quantitative detection of S100B protein in serum and to evaluate its performance.Methods The test strip was prepared using time-resolved fluorescent microsphere-labeled anti-S100B polyclonal antibody and rabbit IgG antibody,labeling pads,sample pads,S100B nitrocellulose films and absorbent paper,and an S100B time-resolved fluorescence immunoassay kit was obtained by assembling the cartridge.The performance of the kit developed was evaluated by standard curve,accuracy,minimum detection limit,linear interval,specificity,reproducibility and stability.The reference intervals of 199 pieces of healthy human serum and plasma samples from a certain region were detected with the kit,and the clinical performance of the kit and Roche Elecsys S100 kit was tested by synchronous blind method to assess the consistency of the results of the two kits for 142 samples.Results The S100B time-resolved fluorescence immunoassay kit had the standard curve beingy=(1.133 02+1.752 24)/[1+(x/1.082 20)×(-0.603 52)]-1.752 24,R2=0.999 08 and the linear range being[0.05,30]ng/mL,which met the requirements of the relative deviation of the accuracy within±15％,the minimum detection limit not hgier than 0.05 ng/mL,the relative deviation of specificity within±15％and the coefficient of variation of intra-and inter-batch difference less than 15％.The stability test results indicated that the kit was valid for 12 months at 2-30 ℃ conditions.The reference intervals of serum and plasma samples measured by the kit were both lower than 0.3 ng/mL.Clinical trials showed that the results by the kit and Roche Elecsys S100 Assay Kit were in high agreement(Kappa=0.906 1＞0.80)and met the requirements.Conclusion The kit developed detects the concentration of S100B protein in serum quickly,accurately and quantitatively,and provides references for the diagnosis and treatment of neurological diseases,autoimmune diseases,cerebrovascular diseases and etc.[Chinese Medical Equipment Journal,2024,45(1):47-55]

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Peste des petits ruminants virus (PPRV) causes a very devastating disease in sheep and goats. Rapid diagnosis and immunisation have been identified as key strategies for successful prevention of the disease. Therefore, a sensitive fluorescent microsphere immunochromatography test strips (FM-ICTS) was developed for rapid detection of special antibodies of PPRV in goats and sheep serum. The FM-ICTS were successfully prepared by fluorescent microspheres (FM) as tracer, which were covalently coupled to PPRV nucleocapsid protein (NP). The NP and monoclonal antibody of NP were separately dispensed onto a nitrocellulose membrane as test and quality control lines, respectively. The critical threshold for determining negative or positive through the ratio of the fluorescent signal of the test line and the control line (T/C) is 0.050. The repeatability of the FM-ICTS was excellent, with an overall average CV of 3.17 %. The detection limit of this assay was 1:5120. Additionally, the FM-ICTS no cross reaction with the sera of other related diseases was observed, only reacting with anti-PPRV serum. 70 serum samples were tested by FM-ICTS and commercial ELISA kit, and the results showed good agreement. Overall, a promising pen-side diagnostic tool was developed for the rapid qualitatively/semi-quantitatively detection of PPRV antibodies within 15 min.

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Tumor markers play a significant role in early cancer diagnosis, evaluation of the extent of the disease, and monitoring of therapy response. In this study, we described the Pickering emulsion polymerization method to synthesize uniform magnetic/fluorescent microspheres. A Pickering-structure composed of a lot silica nanoparticle closely covered onto the quantum dot-encoded magnetic microbeads is designed and synthesized. The uniform magnetic/fluorescent microspheres were prepared using a microfluidic device and the performance of the microspheres synthesized by the instruments was evaluated by flow cytometry. To avoid fluorescence quenching and intrinsic toxicity, CdSe/ZnS core-shell quantum dot and Fe3O4 nanoparticle were successfully encapsulated into MFM microspheres using the microfluidic technology. Using this structure enables the facile realization of a theoretical 4 × 4 barcoding matrix combining two colors and four fluorescence intensity levels. Then, different optical codes were prepared by simple changing the emission wavelength and the intensity of the quantum dots. The resulting microsphere are combined with flow cytometer using two lasers for decoding of multiplex tumor markers. Moreover, the stability testing of microspheres demonstrated good performance for further application in detection of tumor markers as well. When applied for the high-throughput ultrasensitive detection of three tumor markers (CEA, CA125 and CA199) in a single sample, the detection limits of 0.027 ng/mL for CEA, 1.48 KU/L for CA125 and 1.09 KU/L for CA199 are achieved, which exhibit superior detection performance. Thus, Pickering-structure magnetic/fluorescent microspheres are promising for application in tumor markers.

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Helicobacter pylori (H. pylori) is a kind of microaerobic and food-borne pathogen. More than 4.4 billion individuals have been infected by H. pylori and H. pylori was listed as Group I carcinogen by WHO in 1994. Considering the high infection rate of H. pylori and the limited medical resources, self-testing is helpful for diagnosis and timely treatment. Although the amount of H. pylori in human saliva is low, the sampling of saliva is simple and convenient compared with stomach, blood and stool samples. Therefore, H. pylori in human saliva can be an indicator for self-testing, and a sensitive and easy-to-use assay is necessary. In this study, we developed a time-resolved fluorescent microsphere-lateral flow immunoassay (TRFM-LFIA) strip assay with image visual analysis for detection of H. pylori in saliva. The detection of the TRFM-LFIA strip was easy to use and had a low dependency on equipment. With optimized preparation and detection parameters, the whole detection process could be finished in 8 min and the LOD by naked eyes was 102 CFU/mL. For quantitative analysis by image visual analysis, the LOD was as low as 1.05 CFU/mL in the linear range of 101-105 CFU/mL. Besides, the TRFM-LFIA strip also had good stability, reliability, repeatability and accuracy. All these advantages indicated that the TRFM-LFIA strips developed in this study had a good potential for self-testing for H. pylori infection.

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Thiacloprid (TCL) is a neonicotinoid insecticide. Its widespread use has led to high levels of residue in fruits and vegetables. Hence, it is important to detect TCL rapidly, accurately, and sensitively in fruits and vegetables. Recombinant antibodies (rAbs) can be synthesized rapidly with little batch-to-batch variation. In this study, recombinant single-chain variable fragment (scFv) antibody and full-length recombinant antibody against TCL were produced using three different expression systems (E. coli, yeast, and mammalian cell). The results of SDS-PAGE and non - competitive enzyme-linked immunosorbent assay (ELISA) indicated that the full-length rAb exhibited promising characteristics, and the IC50 value of indirect competitive ELISA (ic-ELISA) was 2.63 µg L-1. However, recombinant scFv antibody had little affinity for the antigen. To understand antibody recognition, the three-dimensional (3D) model of the variable fragment (Fv) was built via homologous modeling. The interaction between Fv and TCL was analyzed via molecular docking and the results of molecular docking showed that VAL-158, ALA-211, PHE-220, TRP-218, TRP-49, and ILE-100 were mainly responsible for antibody recognition. In addition, a time-resolved fluorescent microsphere-immunochromatographic test strip (TRFM-ICTS) was developed with a linear range and limit of detection of 0.01-10 ng mL-1 and 0.003 ng mL-1 within 15 min under optimal conditions. The IC50 value was 4.268 ng mL-1, and the recovery ranged between 79.4% and 118.6%, which was consistent with HPLC-MS. The TRFM-ICTS has great advantages in sensitivity and applicability.

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Despite decades of efforts, we are faced with the daunting task of on-site ultratrace environmental toxins detecting, especially the microcystins caused by water bloom. In this work, a novel fluorescent microsphere-based flow-through immunoaffinity chromatographic assay has been designed for detecting ultratrace microcystin-LR in water and aquatic products. The aggregation-induced emission luminogens were encapsulated into fluorescent microspheres to ensure microcystin-LR quantitation with a whole analytical time of less than 30 min. Furthermore, the colorimetric images were captured and quantitatively analyzed, which offered a limit of detection at 0.217 pg/mL and a limit of quantitation at 0.362 pg/mL in water and aquatic muscle samples. The developed immunoassays provide average recovery ranging from 79.1 % to 95.7 %, with relative standard deviations less than 13.4 %. Thus, the validated flow-through immunoaffinity chromatographic assay is an easy-to-use alternative for on-site screening of microcystin-LR in water and aquatic samples at picogram levels.

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BACKGROUND: Mycotoxins are secondary metabolites produced by fungi, which have serious effects on humans and animals. In this study, we selected the monodispersed polystyrene fluorescent microspheres with good luminescence performance and strong stability as markers to conjugate with four mycotoxins antibodies for preparing fluorescent probes. We have developed a fluorescent microsphere based immunochromatographic assay (FMICA) to detect sensitively and quickly zearalenone (ZEN), aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ), and ochratoxin A (OTA) in cereal. RESULTS: Under optimal experimental conditions, the procedure of this method can be completed within 10 min. The limit of detection (LOD) of FMICA for ZEN, AFB1 , FB1 , and OTA is 0.072, 0.093, 0.32, and 0.19 µg L-1 , respectively. And FMICA has good specificity and no cross-reactivity with other mycotoxins. Four mycotoxins in naturally contaminated cereal samples (corn, rice, and oat) are detected by this method, and the results are highly consistent with that of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). CONCLUSION: The developed FMICA has good accuracy, high sensitivity, simplicity, convenience, rapidity, and low cost, and it could be employed for sensitive and quantitative detecting of mycotoxins in cereal on-site. © 2022 Society of Chemical Industry.

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Florfenicol (FF), used popularly in prevention and treatment of virus infections in livestock and poultry, has widely been found in eggs and harmful to human health. In this work, a sensitive and quantitative on-site detecting solution, monoclonal antibody-based carboxylated fluorescent microsphere immunochromatographic test strip assay (FM-ICTS), is design and applied for FF detection. The proposed method can sensitively detect FF in low detection limit of 0.030 ng/g and quantitatively measure its concentration from 0.1 ng/mL to 8.1 ng/mL (R2 = 0.9991) with high repeatability (CV<8.0 %). In addition, the established FM-ICTS method exhibited high measurement accuracy in FF samples as compared with HPLC-MS analysis and demonstrated satisfied recoveries (99.1-101.3 %). More importantly, the quantitative FF test strip demonstrate ultra-high stability, which presents approximately equivalent detection ability to the fresh one after stored at 4 °C for more than one year or stored at 37 °C for 60 days. Therefore, the proposed method is a promising solution for rapidly and sensitively quantitative determination of FF in eggs.

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The multiplex capabilities of the new xMAP INTELLIFLEX DR-SE flow analyzer were explored by modifying a serological assay previously used to characterize the IgG antibody to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The goal was to examine the instrument's performance and to simultaneously measure IgM and IgG antibody responses against multiple SARS-CoV-2 antigens in a single assay. Specific antibodies against the SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins were investigated in 310 symptomatic case patients using a fluorescent microsphere immunoassay and simultaneous detection of IgM and IgG. Neutralization potential was studied using the addition of soluble angiotensin-converting enzyme 2 (ACE2) to block antibody binding. A profile extending to 180 days from symptom onset (DFSO) was described for antibodies specific to each viral antigen. Generally, IgM levels peaked and declined rapidly ∼3-4 weeks following infection, whereas S- and RBD-specific IgG plateaued at 80 DFSO. ACE2 more effectively prevented IgM and IgG binding in convalescent cases > 30 DFSO, suggesting those antibodies had greater neutralization potential. This work highlighted the multiplex and multi-analyte potential of the xMAP INTELLIFLEX DR-SE, and provided further evidence for antigen-specific IgM and IgG trajectories in acute and convalescent cases. IMPORTANCE The xMAP INTELLIFLEX DR-SE enabled simultaneous and semi-quantitative detection of both IgM and IgG to three different SARS-CoV-2 antigens in a single assay. The assay format is advantageous for rapid and medium-throughput profiling using a small volume of specimen. The xMAP INTELLIFLEX DR-SE technology demonstrated the potential to include numerous SARS-CoV-2 antigens; future work could incorporate multiple spike protein variants in a single assay. This could be an important feature for assessing the serological response to emerging variants of SARS-CoV-2.

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Background: Colorectal cancer (CRC) represents a major health concern that can be screened for by the fecal immunochemical test (FIT), which detects blood in the stool. CRC detection sensitivity for hemoglobin (Hb) combined with transferrin (Tf) is higher than for hemoglobin alone. Methods: We developed a europium fluorescent microsphere-based quantitative lateral flow immunochromatography strip to detect fecal Hb and Tf. Performance was tested using fecal samples from 51 patients with CRC and 122 normal subjects. Test strips were generated using paired mouse anti-human Hb and mouse anti-human Tf monoclonal antibodies and tested using standard Hb and Tf samples. Fluorescence was observed at 365 nm and quantitatively measured using a portable fluorescent strip reader. Results: At cutoff values of 100 ng/mL (10 µg/g feces) and 25 ng/mL (2.5 µg/g feces) for Hb and Tf, respectively, the positive rates for Hb, Tf, and Hb+Tf in normal subjects were 6.56%, 5.74%, and 10.66%, respectively, compared to 88.24%, 64.71%, and 94.12% in patients with CRC. The sensitivity and specificity of the FIT combined detection technique were 87.5% and 89.2%, respectively, and the area under the curve (AUC) was 0.92. The sensitivity, specificity, and AUC for the Tf assay were 63.8%, 68.4%, and 0.759, respectively, and those for Hb testing were 69.7%, 70.2%, and 0.774, respectively. The AUC for Hb+Tf was significantly higher than those for Tf or Hb alone (P < 0.001). Conclusions: Fluorescent microsphere-based immunochromatographic strips sensitively detect fecal Hb and Tf, and sensitivity and specificity are improved for Hb+Tf. This system represents a rapid and portable alternative for on-site early CRC screening.

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The mouse model of multiple cerebral infarctions, established by injecting fluorescent microspheres into the common carotid artery, is a recent development in animal models of cerebral ischemia. To investigate its effectiveness, mouse models of cerebral infarction were created by injecting fluorescent microspheres, 45-53 µm in diameter, into the common carotid artery. Six hours after modeling, fluorescent microspheres were observed directly through a fluorescence stereomicroscope, both on the brain surface and in brain sections. Changes in blood vessels, neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry. The microspheres were distributed mainly in the cerebral cortex, striatum and hippocampus ipsilateral to the side of injection. Microinfarcts were found in the brain regions where the fluorescent microspheres were present. Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia. These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts. This model is an effective, additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions. This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. D2021-03-16-1) on March 16, 2021.

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Three kinds of immunochromatographic assays (ICAs) are proposed for the highly sensitive and rapid determination  of tylosin (TYL) and tilmicosin (TIM) in eggs based on colloidal gold (CG), latex microsphere (LM), and time-resolved fluorescent microsphere (TRFM). Three types of ICAs could tolerate the egg matrix via simple sample pretreatment and demonstrated high sensitivity for TYL and TIM with cut-off values of 6/6/3 µg/kg and 14/14/6 µg/kg, respectively. Furthermore, in a single-blind parallel study 20 egg samples were analyzed  by the three developed ICAs and confirmed by LC-MS/MS. The  results showed good consistency, and there were no false positive and false negative results in our three ICAs. Consequently, the proposed three ICAs offered rapid, highly sensitive, reliable, and selectable testing platforms for screening veterinary medicine or other small molecule contaminants.

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Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

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Examination of cerebrospinal fluid in atypical bacterial meningitis (ABM) is similar to that of viral encephalitis (VE), so ABM can easily be misdiagnosed as VE, which can delay diagnosis and treatment. We developed a simple, rapid hand-held lateral flow immunoassay detection system based on fluorescent microspheres (FMS) for procalcitonin (PCT) detection, which provides an indicator to differentiate between ABM and VE. With this novel method, the antigen-antibody reaction systems involve different species, making the test strips more stable than those utilizing one species. The strips exhibited a wide dynamic range (0.04-50 ng/mL) and good sensitivity (0.03 ng/mL). The function of PCT in the identification of ABM and VE in children was further studied. A significant difference in PCT levels was observed between the ABM and VE groups (P = 0.00) and between the ABM and the normal control groups (P = 0.00). PCT levels were not significantly different between the VE and normal control groups (P = 0.30). The area under the receiver operating characteristic curve of PCT for the diagnosis of ABM was 0.95. These findings collectively indicate the usefulness of the PCT detection method based on FMS for clinically differentiating between ABM and VE.

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From June 11, 2020, a surge in new cases of coronavirus disease 2019 (COVID-19) in the largest wholesale market of Beijing, the Xinfadi Market, leading to a second wave of COVID-19 in Beijing, China. Understanding the transmission modes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the personal behaviors and environmental factors contributing to viral transmission is of utmost important to curb COVID-19 rise. However, currently these are largely unknown in food markets. To this end, we completed field investigations and on-site simulations in areas with relatively high infection rates of COVID-19 at Xinfadi Market. We found that if goods were tainted or personnel in market was infected, normal transaction behaviors between sellers and customers, daily physiological activities, and marketing activities could lead to viral contamination and spread to the surroundings via fomite, droplet or aerosol routes. Environmental factors such as low temperature and high humidity, poor ventilation, and insufficient hygiene facilities and disinfection practices may contribute to viral transmission in Xinfadi Market. In addition, precautionary control strategies were also proposed to effectively reduce the clustering cases of COVID-19 in large-scale wholesale markets.

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