Search Results (643)

Background/Objectives: Miscarriage is an increasingly common event worldwide arising from various factors, and identifying its etiology is important for planning and managing any future pregnancies. It is estimated that about half of early pregnancy loss cases are caused by genetic abnormalities, while a significantly lower rate is found in late pregnancy loss. Multiplex ligation-dependent probe amplification (MLPA) can detect small changes within a gene with precise breakpoints at the level of a single exon. The aim of our study was to identify the rate of copy number variations (CNVs) in spontaneous pregnancy loss samples after having previously tested them via quantitative fluorescence PCR (QF-PCR), with no abnormal findings. Methods: DNA was extracted from product-of-conception tissue samples, followed by the use of an MLPA kit for the detection of 31 microdeletion/microduplication syndromes (SALSA^® MLPA^® Probemix P245 Microdeletion Syndromes-1A, MRC-Holland, Amsterdam, The Netherlands). Results: A total of 11 (13.1%) out of the 84 successfully tested samples showed CNVs. Duplications accounted for 9.5% of the analyzed samples (eight cases), while heterozygous or hemizygous deletions were present in three cases (3.6%). Among all the detected CNVs, only three were certainly pathogenic (3.6%), with two deletions associated with DiGeorge-2 syndrome and Rett syndrome, respectively, and a 2q23.1 microduplication syndrome, all detected in early pregnancy loss samples. For the remaining cases, additional genetic tests (e.g., aCGH/SNP microarray) are required to establish CNV size and gene content and therefore their pathogenicity. Conclusions: MLPA assays seem to have limited value in detecting supplementary chromosomal abnormalities in miscarriages. Full article

Swine infectious diseases, often caused by multiple co-infecting agents, pose severe global threats to pig health and industry economics. Conventional single-plex testing assays, whether relying on pathogen antigens or nucleic acids, exhibit limited efficacy in the face of co-infection events. The modern nucleic acid-based multiplex testing (NAMT) methods demonstrate substantial strengths in the simultaneous detection of multiple pathogens involving co-infections owing to their remarkable sensitivity, exceptional specificity, high-throughput, and short turnaround time. The development, commercialization, and application of NAMT assays in swine infectious disease surveillance would be advantageous for early detection and control of pathogens at the onset of an epidemic, prior to community transmission. Such approaches not only contribute to saving the lives of pigs but also aid pig farmers in mitigating or preventing substantial economic losses resulting from infectious disease outbreaks, thereby alleviating unwanted pressure on animal and human health systems. The current literature review provides an overview of some modern NAMT methods, such as multiplex quantitative real-time PCR, multiplex digital PCR, microarrays, microfluidics, next-generation sequencing, and their applications in the diagnosis of swine infectious diseases. Furthermore, the strengths and weaknesses of these methods were discussed, as well as their future development and application trends in swine disease diagnosis. Full article

Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts. Full article

Background/Objectives: Concurrent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A and B viruses (IAV/IBV), and respiratory syncytial virus (RSV) necessitate rapid and precise differential laboratory diagnostic methods. This study aimed to evaluate the multiplex molecular diagnostic performance of the geneLEAD VIII system (Precision System Science Co., Ltd., Matsudo, Japan), a fully automated sample-to-result precision instrument, in conjunction with the VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (CerTest Biotec, S.L., Zaragoza, Spain). Methods: The specific detection capabilities of SARS-CoV-2, IAV/IBV, and RSV genes were evaluated using virus-spiked saliva and nasal swab samples. Using saliva samples, the viral titer detection limits of geneLEAD/VIASURE and manual referent singleplex RT-qPCR assays were compared. The performance of geneLEAD/VIASURE in analyzing single- and multiple-infection models was scrutinized. The concordance between the geneLEAD/VIASURE and the manual assays was assessed. Results: The geneLEAD/VIASURE successfully detected all the virus genes in the saliva and nasal swab samples despite some differences in the Ct values. The viral titer detection limits in the saliva samples for SARS-CoV-2, IAV, IBV, and RSV using geneLEAD/VIASURE were 10⁰, ≤10⁻², 10⁰, and 10² TCID₅₀/mL, respectively, compared to ≤10⁻¹, ≤10⁰, ≤10⁰, and ≤10⁴ TCID₅₀/mL, respectively, in the manual assays. geneLEAD/VIASURE yielded similar Ct values in the single- and multiple-infection models, with some exceptions noted in the triple-infection models when low titers of RSV were spiked with high titers of the other viruses. The concordance between geneLEAD/VIASURE and the manual assays was high, with Pearson’s R² values of 0.90, 0.85, 0.92, and 0.95 for SARS-CoV-2, IAV, IBV, and RSV, respectively. Conclusions: geneLEAD/VIASURE is a reliable diagnostic tool for detecting SARS-CoV-2, IAV/IBV, and RSV in single- and multiple-infection scenarios. Full article

Mycotoxins are responsible for a multitude of diseases in both humans and animals, resulting in significant medical and economic burdens worldwide. Conventional detection methods, such as enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS), are highly effective, but they are generally confined to laboratory settings. Consequently, there is a growing demand for point-of-care testing (POCT) solutions that are rapid, sensitive, portable, and cost-effective. Lateral flow assays (LFAs) are a pivotal technology in POCT due to their simplicity, rapidity, and ease of use. This review synthesizes data from 78 peer-reviewed studies published between 2015 and 2024, evaluating advances in nanoparticle-based LFAs for detection of singular or multiplex mycotoxin types. Gold nanoparticles (AuNPs) remain the most widely used, due to their favorable optical and surface chemistry; however, significant progress has also been made with silver nanoparticles (AgNPs), magnetic nanoparticles, quantum dots (QDs), nanozymes, and hybrid nanostructures. The integration of multifunctional nanomaterials has enhanced assay sensitivity, specificity, and operational usability, with innovations including smartphone-based readers, signal amplification strategies, and supplementary technologies such as surface-enhanced Raman spectroscopy (SERS). While most singular LFAs achieved moderate sensitivity (0.001–1 ng/mL), only 6% reached ultra-sensitive detection (<0.001 ng/mL), and no significant improvement was evident over time (ρ = −0.162, p = 0.261). In contrast, multiplex assays demonstrated clear performance gains post-2022 (ρ = −0.357, p = 0.0008), largely driven by system-level optimization and advanced nanomaterials. Importantly, the type of sample matrix (e.g., cereals, dairy, feed) did not significantly influence the analytical sensitivity of singular or multiplex lateral LFAs (Kruskal–Wallis p > 0.05), confirming the matrix-independence of these optimized platforms. While analytical challenges remain for complex targets like fumonisins and deoxynivalenol (DON), ongoing innovations in signal amplification, biorecognition chemistry, and assay standardization are driving LFAs toward becoming reliable, ultra-sensitive, and field-deployable platforms for high-throughput mycotoxin screening in global food safety surveillance. Full article

Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella multocida (PM), Mannheimia haemolytica (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes. Full article

Heavy metal ions pose a significant threat to the environment and human health due to their high toxicity and bioaccumulation. Traditional instrumentations, although sensitive, are often complex, costly, and unsuitable for on-site rapid detection of heavy metal ions. Lateral flow assay technology has emerged as a research hotspot due to its rapid, simple, and cost-effective advantages. This review summarizes the applications of lateral flow assay technology based on nucleic acid molecules and antigen–antibody interactions in heavy metal ion detection, focusing on recognition mechanisms such as DNA probes, nucleic acid enzymes, aptamers, and antigen–antibody binding, as well as signal amplification strategies on lateral flow testing strips. By incorporating these advanced technologies, the sensitivity and specificity of lateral flow assays have been significantly improved, enabling highly sensitive detection of various heavy metal ions, including Hg²⁺, Cd²⁺, Pb²⁺, and Cr³⁺. In the future, the development of lateral flow assay technology for detection of heavy metal ions will focus on multiplex detection, optimization of signal amplification strategies, integration with portable devices, and standardization and commercialization. With continuous technological advancements, lateral flow assay technology will play an increasingly important role in environmental monitoring, food safety, and public health. Full article

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. Full article

Background/Objectives: Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy of the biliary tract with limited diagnostic tools for early detection. Current serum markers, such as CA19-9, lack specificity and sensitivity, particularly in early-stage disease, which hinders the effectiveness of curative interventions. This narrative review evaluates the limitations of existing diagnostic approaches and explores the potential of combining liquid biopsy (LB) technologies with CRISPR/Cas-based systems for precise, minimally invasive biomarker detection. Methods: A narrative review was conducted, synthesizing literature from 2018 to 2025 across PubMed, MDPI, Web of Science, Google Scholar, and Embase using MeSH terms such as “cholangiocarcinoma,” “liquid biopsy,” “miRNA,” and “CRISPR/Cas.” Results: Circulating microRNAs (e.g., miR-21, miR-16, miR-877) exhibit high diagnostic accuracy. The RACE (Rolling Circle Amplification-assisted CRISPR/Cas9 Cleavage) platform shows promise for detecting extracellular vesicle (EV)-derived miRNAs with high sensitivity and single-nucleotide specificity. When paired with liquid biopsy, CRISPR-based assays enable real-time, cost-effective, and multiplexed detection of tumor-specific biomarkers. Conclusions: The introduction of LB combined with CRISPR/Cas systems could potentially revolutionize the early and accurate diagnosis of CCA, thereby advancing the overall treatment strategy. However, this method is still under development and requires further testing before it can be incorporated into routine diagnostics. Full article

Staphylococcus aureus is a leading cause of foodborne intoxication globally, driven by its heat-stable enterotoxins (SEs), which pose significant public health risks. This review critically evaluates modern and traditional methodologies for detecting S. aureus and its enterotoxins in food matrices, emphasizing their principles, applications, and limitations. The review includes a dedicated section on sample preparation and pretreatment methods for diverse food substrates, addressing a critical gap in practical applications. Immunological techniques, including ELISA and lateral flow assays, offer rapid on-site screening but face matrix interference and variable sensitivity challenges. Molecular methods, such as PCR and isothermal amplification, provide high specificity and speed for bacterial and toxin gene detection but cannot confirm functional toxin production. Sequencing-based approaches (e.g., WGS and MLST) deliver unparalleled genetic resolution for outbreak tracing but require advanced infrastructure. Emerging biosensor technologies leverage nanomaterials and biorecognition elements for ultra-sensitive real-time detection, although scalability and matrix effects remain hurdles. Mass spectrometry (MALDI-TOF MS) ensures rapid species identification but depends on pre-isolated colonies. Traditional microbiological methods, while foundational, lack the precision and speed of molecular alternatives. The review underscores the necessity of context-driven method selection, balancing speed, sensitivity, and resource availability. Innovations in multiplexing, automation, AI-based methods, and integration of complementary techniques are highlighted as pivotal for advancing food safety surveillance. Standardized validation protocols and improved reporting of performance metrics are urgently needed to enhance cross-method comparability and reliability in outbreak settings. Full article

Celery allergy is a common food allergy, particularly among the European population. Currently, several diagnostic methods are available, including multiplex assays, which are useful for identifying celery-allergic patients. However, all of these methods have certain limitations. Api g 7 is a newly identified celeriac allergen belonging to the defensin protein family. Its clinical relevance lies in the high risk of severe systemic reactions among patients sensitized to this molecule. Patients sensitized to Api g 7 are often co-sensitized to Art v 1, the major mugwort (Artemisia vulgaris) allergen, due to structural similarity between these two molecules. This molecular homology plays a key role in the pathogenesis of celery–mugwort syndrome. Although Api g 7may be a major celery allergen, none of the currently available commercial diagnostic tests are capable of detecting sIgE against it. This highlights the need for the development of new, commercially available diagnostic tools in allergology. Full article

This study explored the distribution and genetic characteristics of respiratory pathogens in outpatients with acute respiratory infections (ARIs) in Yangon, Myanmar, during the 2023 rainy season. Among 267 patients who tested negative for influenza, RSV, and SARS-CoV-2 using rapid diagnostic tests, 84.6% were positive for at least one pathogen according to a multiplex polymerase chain reaction (PCR) assay, the BioFire^® FilmArray^® Respiratory Panel 2.1. The most common viruses detected were rhinovirus/enterovirus (RV/EV) at 37.8%, respiratory syncytial virus (RSV) at 22.4%, and human metapneumovirus (hMPV) at 10.0%. These pathogens co-circulated mainly from July to September, with RV/EV consistently predominant. Symptom comparison among RV/EV-, RSV-, and hMPV-infected patients showed similar clinical features, though fever was more common in hMPV cases. Among RV/EV-positive patients, 59.3% had single infections, while 40.7% experienced co-infections, especially with RSV and adenovirus. Genotyping identified 28 types from five species, primarily RV-A and RV-C, which were genetically diverse. One EV-D68 case was also found, emphasizing its potential risk. This study underscores the genetic diversity and clinical impact of RV/EV and stresses the importance of ongoing molecular surveillance in Myanmar’s post-COVID-19 context to inform effective public health responses. Full article

Piroplasmids (Babesia spp., Rangelia spp., Theileria spp., Cytauxzoon spp.) are tick-borne apicomplexan protozoa that infect, depending on the species, erythrocytes and leucocytes in a wide variety of mammals and birds. The genera Bartonella and Borrelia include vector-borne bacteria that can infect and cause disease in both animals and humans. Detection of hemotropic bacteria and piroplasmids in wild animals is often challenging due to low bacteremia or parasitemia. Digital (d)PCR has proven to be an effective modality for the detection and quantification of DNA of hemotropic pathogens with low parasitemia. This study compared dPCR results from 366 biological samples from seven different Brazilian wild animal groups (5 Xenarthra species, 5 deer species, 3 felid species, 1 canid species, 3 rodent species, 1 bat species, 1 tapir species, and 12 bird species) to two other molecular diagnostic techniques: quantitative real-time (qPCR) and nested (nPCR). For this study, DNA extracted from wild animal blood and spleen samples were subjected to a multiplex dPCR assay for piroplasmids, Bartonella spp., and Borrelia spp. For comparison, the same primers and probes for each agent were used in qPCR assays. Additionally, an nPCR based on the 18S rRNA gene for piroplasmids was performed. The proportions of positive results obtained using dPCR were 85.5% for piroplasmids, 33.6% for Bartonella spp., and 16.7% for Borrelia spp. For all tested agents, dPCR proved to be the technique with the highest sensitivity, making it a useful tool for screening vector-borne agents in biological samples from wild animals with low parasitemia. Full article

Duck enteritis virus (DEV), goose parvovirus (GPV), and muscovy duck parvovirus (MDPV) all have similar symptoms after infection, such as severe diarrhea, which seriously affects the healthy development of the waterfowl industry. Hence, it is important to devise a rapid and precise assay for the detection of these three viruses. In this study, a TaqMan probe-based multi-quantitative polymerase chain reaction (qPCR) assay was developed and optimized. Three specific primers and probes were designed according to the conserved regions of UL6 of DEV, REP of GPV, and VP1 of MDPV, respectively. DEV demonstrated a detection limit of 11.6 copies, GPV detected a limit of 95 copies, and MDPV showcased a detection limit of 14.8 copies. The correlation coefficient is greater than 0.99, and the amplification efficiency is 89% to 93%. These results indicate that the multiplex qPCR assay has high sensitivity, specificity, and stability. Of the 215 clinical samples used in this study, 33 tested DEV positive, 25 tested GPV positive, and 24 tested MDPV positive. Overall, the assay established in the current study presents a rapid, efficient, specific, and sensitive tool for of detecting DEV, GPV, and MDPV. Full article

CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls. Full article