Development of a Gold Nanoparticle-Based ELISA for Detection of PCV2.

In this new methodology, plasmonic ELISA (pELISA) was used to detect Circovirus porcine2 (PCV2) in serum samples without the need for plate reading equipment. This process occurs by adapting the conventional ELISA test with gold nanoparticles (AuNPs) to promote a color change on the plate and quickly identify this difference with the naked eye, generating a dark purple-gray hue when the samples are positive and red when the samples are negative. The technique demonstrated high efficiency in detecting samples with a viral load ≥ 5 log10 copies/mL. Plasmonic ELISA offers user-friendly, cost-effective, and reliable characteristics, making it a valuable tool for PCV2 diagnosis and potentially adaptable for other pathogen detection applications.

Comprehensive Profiling and Therapeutic Insights into Differentially Expressed Genes in Hepatocellular Carcinoma.

Background: Drug repurposing is a strategy that complements the conventional approach of developing new drugs. Hepatocellular carcinoma (HCC) is a highly prevalent type of liver cancer, necessitating an in-depth understanding of the underlying molecular alterations for improved treatment. Methods: We searched for a vast array of microarray experiments in addition to RNA-seq data. Through rigorous filtering processes, we have identified highly representative differentially expressed genes (DEGs) between tumor and non-tumor liver tissues and identified a distinct class of possible new candidate drugs. Results: Functional enrichment analysis revealed distinct biological processes associated with metal ions, including zinc, cadmium, and copper, potentially implicating chronic metal ion exposure in tumorigenesis. Conversely, up-regulated genes are associated with mitotic events and kinase activities, aligning with the relevance of kinases in HCC. To unravel the regulatory networks governing these DEGs, we employed topological analysis methods, identifying 25 hub genes and their regulatory transcription factors. In the pursuit of potential therapeutic options, we explored drug repurposing strategies based on computational approaches, analyzing their potential to reverse the expression patterns of key genes, including AURKA, CCNB1, CDK1, RRM2, and TOP2A. Potential therapeutic chemicals are alvocidib, AT-7519, kenpaullone, PHA-793887, JNJ-7706621, danusertibe, doxorubicin and analogues, mitoxantrone, podofilox, teniposide, and amonafide. Conclusion: This multi-omic study offers a comprehensive view of DEGs in HCC, shedding light on potential therapeutic targets and drug repurposing opportunities.

Colorimetric Kit for Rapid Porcine Circovirus 2 (PCV-2) Diagnosis.

The aim of the current study is to present a low-cost and easy-to-interpret colorimetric kit used to diagnose porcine circovirus 2 (PCV-2) to the naked eye, without any specific equipment. The aforementioned kit used as base hybrid nanoparticles resulting from the merge of surface active maghemite nanoparticles and gold nanoparticles, based on the deposition of specific PCV-2 antibodies on their surface through covalent bonds. In total, 10 negative and 40 positive samples (≥102 DNA copies/µL of serum) confirmed by qPCR technique were tested. PCV-1 virus, adenovirus, and parvovirus samples were tested as interferents to rule out likely false-positive results. Positive samples showed purple color when they were added to the complex, whereas negative samples showed red color; they were visible to the naked eye. The entire color-change process took place approximately 1 min after the analyzed samples were added to the complex. They were tested at different dilutions, namely pure, 1:10, 1:100, 1:1000, and 1:10,000. Localized surface plasmon resonance (LSPR) and transmission electron microscopy (TEM) images were generated to validate the experiment. This new real-time PCV-2 diagnostic methodology emerged as simple and economic alternative to traditional tests since the final price of the kit is USD 4.00.

Fast and reliable detection of SARS-CoV-2 antibodies based on surface plasmon resonance.

Researchers worldwide have been studying alternatives to detect SARS-CoV-2 (COVID-19), and accurate and timely diagnosis is crucial for controlling the outbreaks of the disease. Surface plasmon resonance (SPR) is an effective strategy based on antibodies, and it can be used for simple and fast detection of antibodies due to COVID-19 infection. Accordingly, this paper reports on the highly sensitive and specific detection of antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in COVID-19 patients. In this methodology, spike (S) and nucleocapsid (N) proteins belonging to the coronavirus genome were immobilized on the surface of a gold sensor using self-assembled monolayers. Previously, serum from COVID-19 patients was screened by immunochromatography-based COVID-19 IgG rapid test and/or ELISA in house to determine the presence of IgG titers. Serum from COVID-19-positive patients presenting with IgG were added on the surface and, at the time they bound to proteins, they caused refractive changes in the SPR angle. The antibody detection limit was determined through successive injections into the SPR apparatus - these injections ranged from pure (without dilution) to 1 : 200 µL. The system has shown good reproducibility between runs after coated surface regeneration with 0.1 M glycine-HCl solution (pH 3.0); all experiments were tested in triplicate. The antibodies targeted both S and N fragments and gave a high assay sensitivity by identifying 19 out of 20 COVID-19-positive patients. Most importantly, the assay time took less than 10 min. The results of this study indicate that the proposed simple strategy demonstrates high sensitivity and time-saving in the detection of SARS-CoV-2 response antibodies.

A Study of the Effects of pH and Surfactant Addition on Gold Nanoparticle Aggregation.

Gold nanoparticles (AuNPs) can provide a simple, easy-to-use, inexpensive, at hand point-of-care (POC) fast to diagnose; however, AuNPs have the predisposition to form aggregations. Since the nanoparticles stability is an important issue, this article is aiming to study the long-term stability associated with the development of an immunosensor for clinical diagnosis. Here, we assessed two previous methods commonly described in the literature to prevent the formation of aggregate by studying pH and Tween® 20 (polysorbates) addition as surfactant. AuNPs were characterized through ultraviolet-visible (UV-Vis), dynamic light scattering (DLS) and transmission electron microscopy (TEM) and through analysis in the ImageJ software. We found that the Tween® 20 provided more than stable condition in aqueous solution in comparison to pH dependence. The fabricated AuNPs were further used to detect dengue virus and demonstrating that its use at pH 7.2 did not maintain reproducibility in the detection of dengue virus after one year. Unlike, the Tween® 20 modified AuNPs that detected dengue virus soon after the synthesis and over the course of one year demonstrating the high sensitivity of immunosensor. Finally, our result showed excellent dispersity throughout the year when using Tween® 20 to avoid aggregation.

Canine distemper virus detection by different methods of One-Step RT-qPCR / Detecção do vírus da cinomose canina por diferentes métodos de One-Step RT-qPCR

ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

RESUMO: Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para avaliar a sensibilidade analítica dos sistemas A e B. Na reação da curva padrão com RNA sintético, o limite de detecção do sistema A foi de 11 cópias de RNA µL-1. No sistema B foi de 110 cópias de RNA µL-1 na One-Step RT-qPCR e 11 cópias de RNA µL-1 na NESTED-qPCR. A relação entre os valores de Ct e concentração de RNA foi linear. O RNA extraído das diluições da urina foi detectado nas diluições de 10-3 e10-2 pelos sistemas A e B, respectivamente. A centrifugação prévia da urina aumentou a sensibilidade analítica da análise e mostrou ser importante para a rotina diagnóstica. A reação de One-Step RT-PCR é um método rápido, sensível, específico e aplicável na rotina de diagnóstico molecular da cinomose e em projeto de pesquisa que requer metodologia quantitativa e sensível.