Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-β genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish.

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (T_a) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.

Pork adulteration is a major concern for Muslims and Jews whose diets are restricted by religious beliefs, as well as those who are allergic to pork meat and its derivatives. Accurate pork authentication is of great importance to assist this demographic group of people in making decision on their product purchase. The aim of this study was to develop a new analytical method for pork authentication in processed-meat products based on a combination of loop-mediated isothermal amplification (LAMP) and AuNP-nanoprobe colourimetric assay. The LAMP conditions were first optimised to obtain the highest yield of amplified DNA products within the shortest time. Oligoprobe-functionalised AuNPs were then hybridised with LAMP-DNA amplicons, and subsequently challenged with MgSO ₄ at a high concentration to induce AuNP aggregation. In the presence of pork DNA, the colloidal AuNPs-probe remained unchanged in its red colour, which indicates the dispersion of AuNPs. In contrast, in the absence of pork DNA, the colour was changed to colourless as a result from the aggregation of AuNPs. The LAMP-AuNP-nanoprobe assay offers a high sensitivity with a limit of detection as low as 100 pg of pork DNA. The assay is highly specific to pork content without cross-reactivity with the other meat species tested. The assay developed herein can become a simple, inexpensive, precise, and rapid analytical tool for small laboratories or the general public interested in halal food authentication.

Infection with human immunodeficiency virus (HIV)-1 leads to acquired immunodeficiency syndrome (AIDS) if left untreated. According to UN figures, approximately 39 million people globally were living with HIV in 2022, with 76% of those individuals accessing antiretroviral therapy. Measurement of plasma viral RNA load using calibrated nucleic acid amplification tests (like reverse transcription quantitative PCR, RT-qPCR) is routinely performed to monitor response to treatment and ultimately prevent viral transmission. RNA quantities measured by commercial tests can vary over many orders of magnitude, from trace single copy levels to, in cases, over 10 ⁹ /mL of plasma, presenting an analytical challenge for calibrating across a broad measurement range. Interlaboratory study CCQM-P199 “HIV-1 RNA copy number quantification” (April to September 2019) was conducted under the auspices of the Consultative Committee for Amount of Substance (CCQM) Nucleic Acid analysis Working Group (NAWG), with the aims of supporting national metrology institutes (NMIs) and designated institutes (DIs) development of the capacity and evaluating candidate reference measurement procedures for applied viral nucleic acid measurements. Thirteen laboratories participated in CCQM-P199 and were requested to report the RNA copy number concentration, expressed in copies per microliter, of the HIV-1 group specific antigen ( gag ) gene of in vitro transcribed RNA molecules at low (≈ 10 ³ /μL) and high concentration (≈ 10 ⁹ /μL) (Study Materials 1 and 2, linked by gravimetric dilution) and purified genomic RNA from cultured virus (Study Material 3). Study Materials 1 and 3 were measured by participants using one-step reverse transcription digital PCR (RT-dPCR) (Bio-Rad reagents) and/or two-step RT-dPCR with alternative cDNA synthesis reagents. Study Material 2 was measured by both RT-dPCR (one-step) ( n = 4) and orthogonal methods: single molecule flow cytometric counting ( n = 2), high performance liquid chromatograph (HPLC) ( n = 1) and isotype dilution-mass spectrometry (ID-MS) ( n = 1). Interlaboratory reproducibilities (expressed as %CV) were 21.4 %, 15.3 % and 22.0 % for Study Materials 1, 2 and 3 respectively. Analysis of overdispersion showed that the interlaboratory variation for all three Study Materials was not accounted for in their reported uncertainties, indicating uncharacterized sources of variation remain. Although the mean values of RT-dPCR and orthogonal method results were not statistically significantly different ( p = 0.46), the extrapolated mean Study Material 2 results were higher than mean Study Material 1 results (1196 vs . 808 /μL; p < 0.05). Follow-up analysis of Study Material 2 purity by ultra-performance liquid chromatography (UPLC) indicated higher molecular weight (MW) impurities constituted 16.6 % of the molecules, which are hypothesised to be the cause of the HPLC and ID-MS results being higher than the majority of Study Material 1 and 2 results. This study demonstrates that reproducible measurement of RNA templates was achieved by metrology laboratories, illustrating the potential of RT-dPCR combined with complimentary orthogonal approaches to support traceability and precision of contemporary methods for RNA quantification. This study also highlighted that detailed characterization of RNA materials and sources of bias affecting measurements such as RT efficiency is needed to further establish RT-dPCR as a primary reference measurement procedure for RNA copy number quantification.