Search Results (407)

Boro II (BT), the first cytoplasmic male sterility (CMS) system in rice, is widely used in three-line japonica hybrid rice production. Accurate detection of maintainer-seed contamination in BT-type CMS seed lots is critical for ensuring genetic purity and hybrid seed quality. In this study, we developed a SYBR Green-based quantitative real-time PCR (qPCR) assay for the detection and quantification of maintainer-seed contamination in BT-type CMS seed lots. Maintainer-specific primers targeting a mitochondrial sequence unique to the maintainer line, together with an endogenous reference targeting a conserved mitochondrial sequence present in both maintainer and CMS lines, were validated for specificity. A standard curve was constructed using defined CMS–maintainer seed mixtures (0.1–5% contamination), and ΔCt values were converted to relative abundance (2^−ΔCt). The assay exhibited high specificity, reproducibility, and sensitivity, with a strong linear relationship between 2^−ΔCt values and actual contamination levels (R² > 0.99). Performance testing using simulated contamination samples (0.2–3.13%) demonstrated accurate quantification with acceptable recovery rates. This method provides a rapid, robust, and reliable tool for routine genetic purity testing and quality control in BT-type CMS hybrid rice seed production. Full article

Background/Objectives: Immune-inflammatory activation is a central feature of aneurysmal subarachnoid hemorrhage (aSAH), yet the epitranscriptomic mechanisms underlying this response remain insufficiently understood. This study aimed to investigate RNA methylation-associated immune dysregulation in aSAH and to identify potential biomarkers and signaling pathways. Methods: Four Gene Expression Omnibus datasets were analyzed to characterize RNA methylation regulator-related immune alterations in aSAH. Single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), and intersection with ImmPort immune genes were used to identify candidate genes. A total of 159 machine learning combinations were evaluated for model construction and external validation. Two-sample Mendelian randomization, single-cell RNA sequencing (scRNA-seq), and CellChat analyses were further performed. Peripheral blood samples from patients with aSAH (n = 12) and matched healthy controls (n = 12) were used for total m6A quantification and quantitative real-time PCR (qRT-PCR) validation, while Western blotting and immunofluorescence were used to validate the protein expression of LIFR, GP130, IGF2BP2, and RBM15B. Results: Eleven RNA methylation regulators were differentially expressed between aSAH and controls in GSE122897. The WGCNA module most strongly associated with RNA methylation regulator-related scores was enriched in immune response and myeloid activation pathways. Intersection analysis identified 25 candidate immune-inflammatory genes associated with RNA methylation regulator-related transcriptional patterns. Among 159 algorithms, an XGBoost-LASSO pipeline selected oncostatin M (OSM) as the key variable, and the resulting RNA methylation regulator-related immune-derived gene signature (RMRIGS) showed good discrimination between aSAH and controls across training and validation cohorts. Mendelian randomization supported a protective association of genetically predicted OSM expression with subarachnoid hemorrhage risk (IVW OR = 0.66, p = 0.014). Single-cell analysis showed that Osm was predominantly enriched in infiltrating Ccr2+ macrophages, whereas Lifr and Il6st were broadly expressed in activated microglial subpopulations, indicating the presence of an Osm − (Lifr + Il6st) communication axis after SAH. Clinically, total m6A levels were increased in peripheral blood samples from patients with aSAH, and OSM, together with several RNA methylation regulators, was upregulated and associated with m6A-related changes. In experimental models, the protein expression levels of LIFR, GP130, IGF2BP2, and RBM15B were all increased after SAH-related stimulation. Conclusions: RNA methylation programs may be involved in immune dysregulation in aSAH. The OSM-centered RMRIGS was associated with disease status and may provide insight into the interaction between peripheral immune activation and post-SAH neuroinflammation. The potential involvement of the OSM–LIFR/GP130 signaling axis and its association with RNA methylation regulator-related alterations warrant further investigation. Full article

Objectives: This study aimed to evaluate very low HBV DNA viral load below the limit of quantification and to identify correlational factors in different patient groups, including individuals with chronic hepatitis B (CHB), occult HBV infection (OBI), and others. Methods: We retrospectively analyzed 390 patients with very low-level viremia (VLLV). HBV DNA levels were measured in plasma samples using real-time quantitative PCR (qPCR). Serological markers were evaluated in serum samples using chemiluminescence microparticle immunoassay (CMIA). Demographic variables, HBV serological markers (anti-HBs, anti-HBe, anti-HBc), and DNA results were evaluated. Results: The study included 193 CHB patients with maintained virological suppression and 197 patients in the other group; of which, 60 patients had occult hepatitis B infection (HBV DNA positive, HBsAg negative) and 137 had no occult hepatitis B infection. Very low viral load was more common in men (53.3%) and in individuals aged ≥50 years (63.3%). In univariate analysis, OBI was associated with anti-HBe (odds ratio (OR) = 2.874, 95% CI: 1.255–6.579, p = 0.013), and anti-HBc seropositivity (OR = 5.750; 95% CI: 2.626–12.591, p < 0.001). In multivariate analysis, anti-HBe positivity and anti-HBc positivity were independently associated with OBI. Anti-HBs positivity was independently and inversely associated with OBI. Conclusions: In patients with VLLV cohort, anti-HBc and anti-HBe seropositivity were independently associated with detectable but unquantifiable HBV DNA. Although anti-HBe positivity reflects reduced viral replication, it does not indicate complete viral suppression and may be detected at very low viremia levels, especially in occult HBV infection. These findings highlight the complex interplay between viral replication dynamics and host immune responses across the VLLV spectrum, characterize the serological landscape associated with detectable but unquantifiable HBV DNA, and warrant validation in prospective studies. Full article

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we developed and validated an in-house quantitative PCR (qPCR) assay targeting ORF26, optimized for whole blood. Assay calibration used plasmid, BCBL-1 cell–derived, and commercial HHV-8 DNA standards. Analytical validation was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and showed a 95% limit of detection of 65.7 copies/reaction, efficiencies of 90–101% (R² > 0.99), and intra/inter-assay coefficients of variation < 6.5%. Strong correlations were observed among the three calibrators (R² > 0.97).Clinical validation against a composite reference yielded 100% sensitivity, specificity, PPV, and NPV. Viral loads (log₁₀ copies/mL) varied by clinical condition: classic KS and transplant-associated KS showed the lowest medians (2.30–2.23), MCD HIV− and PEL intermediate values (2.83–3.72), and epidemic KS, MCD HIV+, and IRIS-KS the highest (4.12, 4.86, and 5.03, respectively). Viremia > 5 log₁₀ copies/mL was associated with uncontrolled E-KS, MCD HIV+, and IRIS-KS. Longitudinal follow-up revealed viral load decline paralleled clinical improvement. This validated assay provides a robust, affordable tool for HHV-8 quantification in whole blood and supports its integration into diagnostic workflows and patient monitoring. Full article

Background and Aims: Anemia is seen in nearly >70% of patients with cirrhosis and is often non-responsive to nutritional supplements; therefore, we assessed the erythropoiesis and associated alteration in bone marrow (BM). Methods: It is a cross-sectional study. Flow cytometry was performed to assess the hematopoietic stem cells (HSCs) and erythroid population of 60 patients with cirrhosis compared with patients with 7 non-cirrhotic portal fibrosis (NCPF) and 3 controls. Proteomics were performed of the pure CD71 erythroid population taken from patients with cirrhosis to decipher the internal abnormalities supported by validation experiments. Real Time PCR, colony assay and heme quantification, cytokine array, and ELISA were performed to assess erythropoietic stimulating agents (ESA), inflammatory cytokines, and growth factors as an external factor affecting erythropoiesis. Results: We found a decrease in intermediate erythroid progenitors [IEPs; CD71+ CD235a+], conversely early erythroid precursors [EEP; CD71+ CD235a−] and late erythroid progenitors [LEP; CD71− CD235a+] were increased (p < 0.05) in cirrhotic and NCPF as compared to control. However, unlike NCPF, cirrhosis exhibited decreased CD71+ transferrin receptor (TfR1) expression over erythroid cells and increased immature erythrocytes (p < 0.05) in peripheral circulation. In vitro culture of erythroid precursors showed impaired differentiation and maturation that was confirmed by the reduced (p < 0.05) number of erythroid colonies (BFU-E). Proteomics analysis showed downregulated proteins associated with hemoglobin synthesis, ROS detoxification, translation, and mitochondrial activity. Furthermore, we found an altered expression of genes related to erythropoiesis and hemoglobin synthesis and increase (p < 0.05) in inflammatory cytokines such as IL-5, TRAIL-R2, TGF-α, and TGF-β in BM. Conclusions: This study suggests that the dysregulated erythropoiesis observed in patients with cirrhosis having anemia is maintained despite adequate nutrition. Full article

Ginseng Alternaria leaf and stem blight, caused by Alternaria panax, imposes substantial yield and economic losses to the ginseng cultivation industry. Current diagnostic methods for ginseng diseases primarily rely on pathogen isolation from infected tissues, a procedure that is laborious, time-consuming, and inherently low in sensitivity. This study has therefore developed a rapid, specific and sensitive SYBR Green-based quantitative real-time PCR (qPCR) assay for detecting A. panax in plants, seeds, and soil. The developed qPCR assay exhibited high sensitivity and repeatability, with a detection limit of 0.074 fg/μL of target amplicon DNA (0.619 ng/μL of genomic DNA) and a coefficient of variation below 2%. In artificially inoculated tissues (leaves, stems and seeds), Ct values decreased progressively with increasing incubation time, reflecting pathogen proliferation. Analysis of field-collected leaves and stems showed a strong overall correlation between Ct values and visual disease grades. Surveying of ginseng-growing areas revealed that A. panax was detected in asymptomatic leaves and stems at rates of 12.12% and 14.29%, respectively, and in 14.46% of soil samples and 23.73% of seed samples. This qPCR assay presented here provides a robust tool for forecasting early disease, tracking the primary inoculum of the pathogen and its transmission chains, and screening of both ginseng seed lots and candidate soils for ginseng Alternaria leaf and stem blight prior to planting. Full article

Background: Respiratory syncytial virus (RSV) is a leading global pathogen of acute lower respiratory tract infection, posing significant risks to infants, the elderly, and immunocompromised patients. Artemisia argyi Levl.et Vant Extract (AALE) and its active components have a variety of pharmacological effects, but their anti-RSV potential remains unclear. The aim of this study is to investigate the anti-RSV activity of AALE and parthenolide and its underlying mechanisms. Methods: Cell counting kit-8 (CCK-8) assay was used to determine the anti-RSV activities of AALE and parthenolide. Time-of-addition assay and phase of action analysis were used to explore the effect of drugs on the viral replication cycle. Quantitative polymerase chain reaction (qRCR), immunofluorescence (IF) and Western blot (WB) were used to investigate the effects of AALE and parthenolide on RSV-F gene and protein and on RIG-I/TLR-3 pathway related molecules in vitro. In vivo antiviral efficacy was verified by hematoxylin–eosin (HE) staining for lung histopathology, quantitative real-time PCR (qPCR) quantification of RSV-F, RIG-I, TLR-3, IRF3, IL-6, and IFN-β gene expression in lung tissues, and enzyme-linked immunosorbent assay (ELISA) for serum IL-6 and IFN-β levels. Results: AALE exhibited the strongest anti-RSV activity among the extracts (SI = 27.6), while parthenolide was the most potent monomeric compound (SI = 8.19). In vitro, both AALE and parthenolide were effective in the co-treatment and post-treatment models, reducing RSV-F gene and F protein levels in infected cells. Furthermore, they alleviated RSV infection by regulating RIG-I and TLR-3 pathway-related genes and proteins. In vivo, AALE and parthenolide suppressed lung index and RSV proliferation, attenuated lung injury, and down-regulated RIG-I, TLR-3, IRF3, IL-6, and IFN-β expression in the lungs of RSV-infected mice. Conclusions: AALE and its component parthenolide can inhibit the invasion and replication of RSV, making it a potential candidate for the treatment of RSV-related diseases. Full article

In the European Union, mandatory labeling of food and feed products is required when authorized genetically modified organisms (GMOs) exceed 0.9% per ingredient, necessitating reliable analytical methods for official control laboratories. Event-specific PCR assays validated according to ISO/IEC 17025 are the reference approach for GMO detection, identification, and quantification. The growing use of digital PCR (dPCR) has encouraged the adaptation of real-time PCR methods to dPCR-based strategies, as dPCR enables absolute quantification without calibration standards, shows reduced sensitivity to inhibitors, and allows for the design of a multiplex assay. In this study, an in-house validation of a duplex dPCR assay targeting the maize GM event NK603 and the HMG reference gene was performed on three platforms: Bio-Rad QX200™ (Pleasanton, CA, USA), Qiagen QIAcuity (Venlo, The Netherlands), and Thermo Fisher QuantStudio Absolute Q (Waltham, MA, USA). All validation parameters met the Joint Research Centre (JRC) acceptance criteria. In particular, this assay demonstrated high specificity, sensitivity (limit of quantification or LOQ < 35 copies per reaction), precision, and trueness (RSDr and bias <25%). The data indicate that the duplex dPCR assay can be used for routine GMO analysis and future collaborative validation studies. Full article

Background: This study evaluated the inter-method agreement of an in-house qualitative CMV real-time PCR assay for the detection of cytomegalovirus (CMV) DNA in various non-plasma clinical specimen types, in comparison with a commercially available comparator assay. Methods: In this prospective comparative study, 186 clinical specimens—including bronchoalveolar lavage fluid (BALF), stool, urine, colonoscopic biopsy, amniotic fluid, and intraocular fluid—were analyzed. A total of 166 samples with valid results from both test systems were included in the inter-method comparison. CMV DNA was detected using the in-house qualitative PCR assay in parallel with the comparator assay (artus^® CMV QS-RGQ kit). Agreement was assessed using positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA), and Cohen’s kappa coefficient (κ), in accordance with CLSI EP12-A2 recommendations. Results: Substantial overall inter-method agreement was observed when all specimens were evaluated collectively (κ = 0.66). Agreement metrics were highest in stool, urine, and invasive specimens, whereas BALF samples demonstrated comparatively lower agreement, reflecting potential matrix-related analytical variability. Conclusion: The laboratory-developed qualitative CMV PCR assay demonstrated substantial inter-method agreement with the comparator assay across multiple non-plasma specimen types. The findings highlight specimen-specific variability in qualitative CMV DNA detection and represent analytical concordance between two molecular assays rather than definitive clinical diagnostic accuracy or viral load quantification. Full article

Atractylodes macrocephala (A.M.) is a traditional Chinese medicinal and edible herb renowned for its spleen-tonifying, dampness-resolving, diuretic, and antiperspirant properties. Its primary bioactive constituents are terpenoids, which have demonstrated anti-inflammatory, antitumor, and immunomodulatory activities. However, transcriptomic studies focusing on terpenoid biosynthesis in A.M. from different geographical origins remain limited. To investigate the molecular mechanisms underlying differential sesquiterpenoid production, we performed transcriptome sequencing on samples collected from four distinct regions in China. Sesquiterpenoid biosynthesis predominantly proceeds through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. Comparative analysis revealed four key enzyme-encoding genes—HMGCR, ISPF, GCPE, and FDPS—whose differential expression patterns were further validated by quantitative real-time PCR (qRT-PCR). Samples from Shaanxi exhibited the highest upregulation of biosynthetic genes and the greatest enrichment of terpene-related metabolites, suggesting enhanced pharmacological potential. In contrast, samples from Fujian, Anhui, and Hebei displayed relatively lower activity, with only FDPS upregulated in the Hebei sample. High-performance liquid chromatography (HPLC) quantification confirmed regional differences in the levels of major terpenoids—including atractylodin, atractylenolide I, and atractylenolide III—which correlated well with the observed gene expression profiles. This study compared conspecific A.M. from different geographical regions and further revealed that the variation in terpenoid metabolites is closely related to environmental factors. These findings provide a theoretical basis for the further discovery of functional genes and offer important implications for the quality control of A.M. Full article

Background: low-quantity or degraded samples are often studied in forensic genetics. Therefore, it is important to efficiently obtain all the available DNA from the biological sample analyzed to provide the most reliable results. This is particularly challenging in bone marrow processing due to its hydrophobic molecular structure, as for other lipid-rich tissues, especially if rancid. In fact, during adipose tissue decomposition, the putrefaction of fatty acids can in some instances give a compact cerous consistency to the lipidic tissue, hardly susceptible to the nucleic acid extraction mechanisms. According to environmental circumstances, this condition is notably observable in submerged bodies or in putrefied bone marrow. Thus, this study is focused on developing an optimized nucleic acids extraction protocol for putrefied bone marrow. Methods: genetic analyses were performed on putrefied yellow bone marrow collected from 20 human femora recovered from bodies in different decomposition stages. The optimized method was developed by integrating additional steps, reagents and time intervals on a silica-based column commercial kit. This strategy was compared in DNA yield to a standard extraction protocol, represented by the same commercial kit, but following the manufacturer’s directions. Both these strategies were tested in nucleic acid isolation efficiency by performing DNA typing, including real-time PCR quantification, Short Tandem Repeats (STR) amplification and fragments analysis steps. The analytical parameters evaluated were allele count, DNA concentration (ng/µL) and Degradation Index (DI). Results: for allele count and DNA concentration parameters, the optimized protocol showed clear and significant qualitative and quantitative improvements compared with the standard protocol, supporting its potential applicability in forensic casework and laying the foundation for future studies. Conclusions: prior to appropriate laboratory internal validation, the optimized protocol can be used for tough lipid-rich tissues processing without the need to purchase a dedicated system and using a same commercial kit routinely adopted for other forensic genetics matrices. Full article

Real-time PCR (qPCR) is today’s definitive quantitative technology in molecular biology and diagnostics. Until 30 years ago, PCR product analyses were generally performed after amplification using gel-based methods. Quantification typically relied on visual inspection or densitometry of end-point products and was therefore relatively unreliable and poorly suited to high-throughput automation. To celebrate real-time PCR’s 30-year anniversary of commercial availability, Professor Stephen Bustin, Guest Editor for the special edition, “Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond,” asked Russell Higuchi to give a historical account on how his idea of real-time PCR was conceived and brought to fruition. Dr. Higuchi then asked his collaborator, Lincoln McBride, who drove the development of the ABI 7700—the high-throughput real-time PCR instrument that gave researchers access to this technology—to co-author this dual memoir. This story is told from the perspectives of the two scientists most directly responsible for making real-time PCR practical and widely accessible. Taking turns, Russell Higuchi describes the conceptual and experimental steps at Cetus and then Roche that led from homogeneous PCR detection to continuous fluorescence monitoring, whilst Lincoln McBride details ABI’s parallel efforts to commercialize Russ’s invention. Together, they trace how experimental insight, engineering constraints, product development, and commercial decision-making shaped the Applied Biosystems 7700 Sequence Detection System and established real-time PCR as a practical and reliable quantitative technology. Their team’s efforts persevered through technological uncertainty and within a complex corporate collaboration. They share key historical documents in their original form. Their accounts show how the 7700 system emerged as the convergence of chemistry, optics, software, and product development. The eventual global reliance on real-time PCR during the COVID-19 pandemic demonstrated, at unprecedented scale, the profound and enduring impact of these early technical and organizational choices. Full article

A liver fluke, Clonorchis sinensis is a representative fish-borne parasite infecting humans, and sensitive detection in fish hosts or aquatic environments is important for monitoring infection sources in endemic areas. Conventional diagnostic methods based on microscopy or conventional PCR often show limited sensitivity, particularly under low-parasite conditions. In this study, we developed a high-sensitive and species-specific molecular marker and established a real-time PCR (qPCR)-based diagnostic method targeting metacercariae isolated from freshwater fish, representing the transmission stage of C. sinensis. Primers and a hydrolysis probe targeting the mitochondrially encoded cytochrome c oxidase 1 (COI) gene were designed, and all primer combinations produced stable amplifications with single melt curves in C. sinensis-positive samples. Among them, one combination was finally selected as the optimal marker due to its high specificity, including validation against mixed trematode samples to confirm species-specific detection. The qPCR assay showed excellent linearity (R² = 0.998), with a detection limit of 10¹ copies per reaction and a quantification limit of 10² copies per reaction. In addition, the assay successfully detected C. sinensis DNA in environmental water samples spiked with metacercariae, demonstrating its applicability to aquatic samples for environmental surveillance purposes. Compared with conventional PCR, the developed qPCR method in this study exhibited markedly improved sensitivity in fish-derived samples. Overall, this qPCR assay provides a robust diagnostic tool for laboratory analysis and has potential utility for environmental DNA-based monitoring of clonorchiasis risk areas within a One Health framework. Full article

Amplification efficiency is one of the key parameters in quantitative real-time PCR, as it directly influences the accuracy of both absolute and relative quantification. Amplification efficiency, the fold increase per cycle, is affected by oligonucleotide design, reaction chemistry, sample and template properties, and instrument performance. Consequently, it differs between samples, assays and experimental runs. Although methods for estimating the amplification efficiency have been available for more than two decades, most published qPCR studies continue to assume equal and ideal efficiency across assays. This simplifying assumption introduces efficiency- and expression-dependent error into relative expression and fold-change analyses, contributing to the poor reproducibility observed in many PCR-based studies. This review examines the role of the amplification efficiency in qPCR quantification; the consequences of ignoring, assuming or misapplying efficiency; and the practical implications for data interpretation. The review also considers the development of efficiency estimation models and their implications for contemporary analytical approaches, including emerging data-driven methods for amplification curve analyses. Full article

Downy mildew of hops represents a serious disease affecting hops production in all growing regions. Disease management is primarily based on the application of fungicides at regular intervals based on a short-term forecasting methodology that is essential for evaluating the occurrence of theoretical infections. To enable a more reliable assessment of the pathogen’s presence in a given area, spore traps capturing airborne Pseudoperonospora humuli sporangia can be utilized. The use of quantitative real-time PCR (qRT-PCR) for the detection of sporangia collected by these traps allows for the elimination of laborious and time-consuming microscopic counting. Among four tested P. humuli-specific nuclear DNA sequences, an effective qRT-PCR detection method was developed based on the c127233.5e3 sequence. This detection approach was used for the quantification of sporangia from volumetric spore trap samples collected in situ under field conditions at three selected localities in Bohemia and Moravia during the 2021–2022 period. The obtained results were compared with the short-term forecasting method of the downy mildew (HDM) weather index (I) based on meteorological data. The overall course of the HDM weather index (I) closely correlated with the occurrence of sporangia: after reaching the maximum HDM weather index (I) value, the highest sporangium detection was observed with a time delay of 1–2 weeks at all the monitored sites. The results corresponded well with data obtained from volumetric spore traps in Germany, and the qRT-PCR method proved to be fully comparable to light microscopy. The combination of volumetric spore traps and qRT-PCR can significantly improve the precision of short-term forecasting systems for P. humuli infection, thereby enabling more efficient fungicide application programs in hops protection and contributing to a better understanding of the pathogen’s dispersal dynamics. Full article