Development and application of a high-sensitivity immunochromatographic test strip for detecting pseudorabies virus.

Introduction: Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput. Methods: This study was designed to produce monoclonal antibodies targeting PRV through immunization of mice using the eukaryotic system to express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips. Results and discussion: The strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336 × 103 copies/µL (the number of viral genome copies per microliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus.

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

Quantum dot-based fluorescence-linked immunosorbent assay for the rapid detection of lomefloxacin in animal-derived foods.

Lomefloxacin (LMF), a third-generation fluoroquinolone antibacterial agent, is often used to treat bacterial and mycoplasma infections. However, due to its prolonged half-life and slow metabolism, it is prone to residues in animal-derived foods, posing a potential food safety risk. Therefore, it is particularly urgent and important to establish a method for detecting lomefloxacin. In this study, direct and indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) based on quantum dots (QDs) was established for the detection of LMF. As for dc-FLISA, the half-maximal inhibitory concentration (IC50) and limit of detection (LOD) were 0.84 ng/mL, 0.04 ng/mL, respectively, the detection ranges from 0.08 to 9.11 ng/mL. The IC50 and LOD of ic-FLISA were 0.43 ng/mL and 0.03 ng/mL, respectively, meanwhile the detection ranges from 0.05 to 3.49 ng/mL. The recoveries of dc-FLISA and ic-FLISA in animal-derived foods (milk, fish, chicken, and honey), ranged from 95.8% to 105.2% and from 96.3% to 103.4%, respectively, with the coefficients of variation less than 8%. These results suggest that the dc-FLISA and ic-FLISA methods, which are based on QD labelling, are highly sensitive and cost-effective, and can be effectively used to detect LMF in animal-derived foods.

Thiolated chitosan encapsulation constituted mucoadhesive nanovaccine confers broad protection against divergent influenza A viruses.

Influenza A virus (IAV) poses a significant threat to human and animal health, necessitating the development of universal influenza vaccines that can effectively activate mucosal immunity. Intranasal immunization has attracted significant attention due to its capacity to induce triple immune responses, including mucosal secretory IgA. However, inducing mucosal immunity through vaccination is challenging due to the self-cleansing nature of the mucosal surface. Thiolated chitosan (TCS) were explored for mucosal vaccine delivery, capitalizing on biocompatibility and bioadhesive properties of chitosan, with thiol modification enhancing mucoadhesive capability. The focus was on developing a universal nanovaccine by utilizing TCS-encapsulated virus-like particles displaying conserved B-cell and T-cell epitopes from M2e and NP proteins of IAV. The optimal conditions for nanoparticle formation were investigated by adjusting the thiol groups content of TCS and the amount of sodium tripolyphosphate. The nanovaccine induced robust immune responses and provided complete protection against IAVs from different species following intranasal immunization. The broad protective effect of nanovaccines can be attributed to the synergistic effect of antibodies and T cells. This study developed a universal intranasal nanovaccine and demonstrated the potential of TCS in the development of mucosal vaccines for respiratory infectious diseases.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies.

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200-350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251-305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262-279. Amino acid 262-279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262-273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

An atypical GdpP enzyme linking cyclic nucleotide metabolism to osmotic tolerance and gene regulation in Mycoplasma bovis.

Nucleotide second messengers play an important role in bacterial adaptation to environmental changes. Recent evidence suggests that some of these regulatory molecular pathways were conserved upon the degenerative evolution of the wall-less mycoplasmas. We have recently reported the occurrence of a phosphodiesterase (PDE) in the ruminant pathogen Mycoplasma bovis, which was involved in c-di-AMP metabolism. In the present study, we demonstrate that the genome of this mycoplasma species encodes a PDE of the GdpP family with atypical DHH domains. Characterization of M. bovis GdpP (MbovGdpP) revealed a multifunctional PDE with unusual nanoRNase and single-stranded DNase activities. The alarmone ppGpp was found unable to inhibit c-di-NMP degradation by MbovGdpP but efficiently blocked its nanoRNase activity. Remarkably, MbovGdpP was found critical for the osmotic tolerance of M. bovis under K+ and Na+ conditions. Transcriptomic analyses further revealed the biological importance of MbovGdpP in tRNA biosynthesis, pyruvate metabolism, and several steps in genetic information processing. This study is an important step in understanding the role of PDE and nucleotide second messengers in the biology of a minimal bacterial pathogen.

Rapid detection of varicella-zoster virus based on an immunochromatographic strip.

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL-1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV.

Label-free electrochemical immunosensor based on staphylococcal protein a and AgNPs-rGO-Nf for sensitive detection of virginiamycin M1.

Virginiamycin (VIR), a feed additive, is used to promote pig and poultry growth. However, it is hazardous to human health. This work described a label-free electrochemical immunosensor based on silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites and staphylococcal protein A (SPA) for the first time to directly detect the residual marker VIR M1. Good catalytic currents for oxygen reduction reaction were apparently obtained after the modification of nanocomposites on gold electrode. Nanocomposites were characterized using UV-Vis, X-ray diffraction (XRD) patterns, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). SPA was targeted to immobilize VIR M1 monoclonal antibody (mAb) by binding to Fc region of antibody. The proposed immunosensor showed a wide linear range from 0.25 ng mL-1 to 100 ng mL-1, providing detection limit (LOD) of 0.18 ng mL-1 of VIR M1. Recovery rates ranged from 92.27% to 98.84%, and relative standard deviation (RSD) was not above 6.6%, indicating the immunosensor could detect VIR M1 in actual samples with high accuracy. The sensor showed good selectivity, reproducibility and stability and could be considered as a potential tool for detection of VIR M1 in feed and animal derived food.

Electrochemical immunosensor for ultrasensitive detection of human papillomaviruse type 16 L1 protein based on Ag@AuNPs-GO/SPA.

Human papillomaviruse type 16 (HPV16) is a high-risk serotype. As the main protective antigen protein, L1 protein is also the target protein for diagnosis. A simple label free electrochemical immunosensor (ECIS) was fabricated for ultrasensitive detection of HPV16 L1 protein in this work. Quasi-spherical Ag@Au core-shell nanoparticles on graphene oxide (Ag@AuNPs-GO) was developed as current response amplifier and characterized by UV-Vis Spectroscopy, Transmission Electron Microscopy and energy dispersive X-ray spectroscopy. Staphylococcal protein A was decorated on the modified electrode and utilized to immobilized the Fc portion of the monoclonal antibody specific for HPV16 L1 protein. Cyclic Voltammetry, Differential Pulse Voltammetry and Electrochemical Impedance Spectroscopy were used to verify the electrochemical performance and interfacial kinetic property. The increased concentration of HPV16 L1 protein led to slow electron transport and linearly decreased differential pulse voltammetry peak current with a detection limit of 0.002 ng mL-1 and a wide linear relationship in the range of 0.005-400 ng mL-1at a regression coefficient (R2) of 0.9948. Furthermore, this ECIS demonstrated acceptable accuracy with good reproducibility, stability and selectivity, suggesting a promising immunological strategy for HPV typing and early screening.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Identification of a novel B-cell epitope of the African swine fever virus p34 protein and development of an indirect ELISA for the detection of serum antibodies.

African swine fever (ASF) is a viral disease caused by the African swine fever virus that can be highly transmitted and lethal in domestic pigs. In the absence of a vaccine, effective diagnosis is critical for minimizing the virus's spread. In recent years, with the decline of African swine fever virus (ASFV) virulence, antibody detection has become an important means of detection. ASFV nucleocapsid protein p34 is a mature hydrolytic product of pp220, which is highly conserved and has a high content in the structural protein of the virus. Prokaryotic cells were chosen to generate highly active and high-yield p34 protein, which was then used as an antigen for producing mouse monoclonal antibodies. The B-cell epitope 202QKELDKLQT210, which was highly conserved and found on the surface of the p34 protein, was first identified by an anti-p34 monoclonal antibody utilizing the peptide scanning technique and visualized in helix. This supported the viability of p34 protein detection even further. In addition, we established an indirect ELISA assay based on p34 to detect ASFV antibodies. The coincidence rate of this method with commercially available kits was shown to be 97.83%. Sensitivity analysis revealed that it could be detected in serum dilution as low as 1:6400, and there was no cross-reaction with other prevalent porcine epidemic diseases classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus 2 (PCV2). In summary, the established ELISA method and anti-P34 monoclonal antibody have demonstrated that the p34 protein has a promising application prospect for the detection of African swine fever antibodies.

Serological survey of SARS-CoV-2 in companion animals in China.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can be transmitted from human to companion animals. The national wide serological surveillance against SARS-CoV-2 was conducted among pet animals, mainly in cats and dogs, 1 year after the first outbreak of COVID-19 in China. All sera were tested for SARS-CoV-2 IgG antibodies using an indirect enzyme linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of spike protein. This late survey takes advantage of the short duration of the serological response in these animals to track recent episode of transmission. A total of 20,592 blood samples were obtained from 25 provinces across 7 geographical regions. The overall seroprevalence of SARS-CoV-2 infections in cats was 0.015% (2/13397; 95% confidence intervals (CI): 0.0, 0.1). The virus infections in cats were only detected in Central (Hubei, 0.375%) and Eastern China (Zhejiang, 0.087%) with a seroprevalence estimated at 0.090 and 0.020%, respectively. In dogs, the seroprevalence of SARS-CoV-2 infections was 0.014% (1/7159; 95% CI: 0.0, 0.1) in the entire nation, seropositive samples were limited to Beijing (0.070%) of Northern China with a prevalence of 0.054%. No seropositive cases were discovered in other geographic regions, nor in other companion animals analyzed in this study. These data reveal the circulation of SARS-CoV-2 in companion animals, although transmission of the virus to domestic cats and dogs is low in China, continuous monitoring is helpful for the better understand of the virus transmission status and the effect on animals.

Establishment of an Immunological Method for Detection of Bluetongue Virus by Fluorescence-Linked Immunosorbent Assay.

Bluetongue (BT) is a severe noncontagious infectious disease that occurs in sheep and wild ruminants but occasionally also in cattle and camels. The worldwide BT pandemic has had a significant impact on global livestock production. Rapid detection helps prevent outbreaks of bluetongue disease. Fluorescence-linked immunosorbent assay (FLISA) labeled with quantum dots (QDs) is typically used for detection due to its high sensitivity. There has been no reported detection of BT virus (BTV) using QD-based fluorescence immunoassays. In this study, monoclonal antibodies (MAbs) against BT were prepared by immunizing BALB/c mice with recombinant VP7 protein. Two MAbs with high sensitivity and specificity were selected as the detection antibody (2F11) and capture antibody (11B7). Then, the detection antibody was coupled with QDs to prepare QD-MAb fluorescence probes. Fluorescence-linked immunosorbent assay is highly specific, detecting only VP7 protein/BTV, and did not show any nonspecific reactions with other reoviruses. The detection limit of VP7 protein was 3.91 ng/mL using fluorescence-linked immunosorbent assay, with a coefficient of variation (CV) of less than 15%. The establishment of rapid, sensitive direct FLISA has potential for bluetongue virus detection and control of BT vaccine quality. IMPORTANCE Bluetongue virus causes the severe infectious disease BT. BTV has many serotypes, and there is no cross-protection among different serotypes. BT is listed as a notifiable animal infectious disease by the World Organisation for Animal Health (OIE) and occurs throughout the world, causing significant economic losses. The establishment of a fast and effective detection method is the key to controlling and preventing this disease. Current methods for detecting BTV mainly include reverse transcription-PCR (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and immunochromatographic strips that are based on antigen-antibody recognition. Immunoassays are most commonly used because of their low cost, high specificity, and fast analysis, making them particularly useful for routine monitoring. These conventional detection strategies for BTV have some drawbacks. Recently, FLISA has been drawing attention due to its sensitivity, which is higher than traditional immunoassays. Fluorescence-linked immunosorbent assays (FLISA) using fluorescent materials as labels overcome ELISA's disadvantage of being time-consuming.

Novel mycoplasma nucleomodulin MbovP475 decreased cell viability by regulating expression of CRYAB and MCF2L2.

Nucleomodulins are secreted bacterial proteins whose molecular targets are located in host cell nuclei. They are gaining attention as critical virulence factors that either modify the epigenetics of host cells or directly regulate host gene expression. Mycoplasma bovis is a major veterinary pathogen that secretes several potential virulence factors. The aim of this study was to determine whether any of their secreted proteins might function as nucleomodulins. After an initial in silico screening, the nuclear localization of the secreted putative lipoprotein MbovP475 of M. bovis was demonstrated in bovine macrophage cell line (BoMac) experimentally infected with M. bovis. Through combined application of ChIP-seq, Electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis, MbovP475 was determined to bind the promoter regions of the cell cycle central regulatory genes CRYAB and MCF2L2. MbovP475 has similar secondary structures with the transcription activator-like effectors (TALEs). Screening of various mutants affecting the potential DNA binding sites indicated that the residues 242NI243 within MbovP475 loop region of the helix-loop-helix domain were essential to its DNA binding activity, thereby contributing to decrease in BoMac cell viability. In conclusion, this is the first report to confirm M. bovis secretes a conserved TALE-like nucleomodulin that binds the promoters of CRYAB and MCF2L2 genes, and subsequently down-regulates their expression and decreases BoMac cell viability. Therefore, this study offers a new understanding of mycoplasma pathogenesis.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein.

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1-S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

A Universal Fluorescent Immunochromatography Assay Based on Quantum Dot Nanoparticles for the Rapid Detection of Specific Antibodies against SARS-CoV-2 Nucleocapsid Protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus' life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 105 with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges.

Development of an Indirect ELISA Kit for Rapid Detection of Varicella-Zoster Virus Antibody by Glycoprotein E.

Varicella-zoster virus (VZV), a highly infectious agent that causes varicella (chickenpox), can also cause zoster (shingles), a disorder that is frequently associated with severe neuralgia. A reliable serological VZV diagnostic assay would be useful for identifying unprotected individuals and for surveilling post-vaccination immunoprotection status. Toward this goal, VZV membrane glycoprotein E (gE), the immunodominant VZV protein, served as target antigen in an indirect ELISA kit developed here to detect anti-VZV antibodies in clinical samples. For target antigen preparation, Chinese hamster ovary (CHO) cells were modified to express and secrete the VZV gE ectodomain, which was subsequently purified and used as coating antigen in an indirect ELISA. Ultimately, the optimal purified gE coating antigen concentration was determined to be 2 µg.ml-1 and the OD450nm detection cutoff value was 0.286. The coefficient of variation (CV) of intra-assay and inter-assay were <10 and 15%, respectively. A comparative test of 66 clinical samples showed that the coincidence rate was 93.9% between the indirect ELISA and a commercial varicella-zoster virus IgG ELISA kit. Thus, the indirect ELISA kit developed here may be useful for achieving rapid, sensitive, and specific detection of anti-VZV antibodies.

Quantum dot-based fluorescence immunosorbent assay for the rapid detection of bacitracin zinc in feed samples.

Bacitracin zinc (BAC), a polypeptide antibiotic, is utilized as a feed additive due to its ability to promote growth in animals. However, the abuse of BAC can lead to a great threat to food safety. Therefore, there is an urgent need to develop a rapid and sensitive detection method. In this study, a monoclonal antibody (mAb) against BAC with excellent sensitivity and specificity was obtained. For the first time, quantum dots (QDs) were conjugated with the prepared mAb against BAC and rabbit anti-mouse antibody to fabricate a direct and an indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) to detect BAC. The IC50 of dc-FLISA and ic-FLISA were 0.28 ng/ml and 0.17 ng/ml, respectively. The limits of detection were 0.0016 ng/ml and 0.001 ng/ml, respectively, and the detection ranges were 0.0016-46.50 ng/ml and 0.001-35.65 ng/ml, respectively. In addition, the recovery rate of the two methods ranged from 93.5% to 112.0%, and the coefficient of variation (CV) was less than 10%. Therefore, the methods developed in this work have the merits of low cost, simple operation, and high sensitivity, which provide an effective analytical tool for BAC residue detection in feed samples.

Rapid Visual Detection of Hepatitis C Virus Using Reverse Transcription Recombinase-Aided Amplification-Lateral Flow Dipstick.

Hepatitis C virus (HCV) infection is a global public health threat. Reaching the World Health Organization's objective for eliminating viral hepatitis by 2030 will require a precise disease diagnosis. While immunoassays and qPCR play a significant role in detecting HCV, rapid and accurate point-of-care testing is important for pathogen identification. This study establishes a reverse transcription recombinase-aided amplification-lateral flow dipstick (RT-RAA-LFD) assay to detect HCV. The intact workflow was completed within 30 min, and the detection limit for synthesized C/E1 plasmid gene-containing plasmid was 10 copies/µl. In addition, the test showed good specificity, with no cross-reactivity observed for hepatitis A virus, hepatitis B virus, HIV, syphilis, and human papillomavirus virus. Using extracted RNAs from 46 anti-HCV antibody-positive samples, RT-RAA-LFD showed 100% positive and negative concordance rates with qPCR. In summary, the RT-RAA-LFD assay established in this study is suitable for the rapid clinical detection of HCV at the community level and in remote areas.

Differential diagnosis of the infection caused by wild-type or CD2v-deleted ASFV strains by quantum dots-based immunochromatographic assay.

African swine fever (ASF), a highly contagious and lethal disease, poses a tremendous threat and burden to the swine industry worldwide. Lack of available vaccines or treatments leaves rapid diagnosis as the key tool to control the disease. Quantum dots (QDs) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In this study, we developed a quantum dots-based fluorescent immunochromatographic assay (QDs-FICA) using CD2v as the diagnosis antigen to detect ASFV antibodies. The titre of the test strip was 1 : 5·12 × 105 . In addition, the strip was highly specific to anti-ASFV serum and had no cross-reaction with CSFV, PPV, PRRSV, PCV-2, PRV and FMDV. Moreover, a comparative test of 71 clinical samples showed that the coincidence rate was 85·92% between the test strip and the commercial ELISA kit (coated with p30, p62 and p72). The QDs-FICA can be used to detect ASFV antibodies, which is meaningful for the surveillance, control and purification of ASF.