Search Results (1,608)

Cymbidium eburneum Lindl. is a valuable ornamental orchid and breeding parent, but its genetic background remains unclear due to habitat destruction and germplasm mixing. This study developed specific SSR markers to evaluate the genetic diversity and structure of 96 C. eburneum Lindl. accessions from China and Vietnam. Transcriptome analysis identified 47,248 SSR loci. Sixteen polymorphic core primer pairs detected 150 alleles (mean Na = 9.375) with an average Polymorphism Information Content (PIC) of 0.444. Observed heterozygosity (Ho = 0.290) was noticeably lower than expected (He = 0.478), indicating heterozygote deficiency. UPGMA clustering identified eight groups strongly correlated with geography. Principal Coordinate Analysis (PCoA) revealed a clear geographical differentiation pattern, featuring the most genetically cohesive group from Guangxi and more differentiated geographically marginal populations from Hainan and Vietnam. STRUCTURE analysis (K = 2) indicated two main gene pools with signals of genetic admixture. Geographical isolation was suggested as a potential driver of genetic differentiation. The Guangxi population represents a genetically consistent major reservoir, while marginal populations harbor unique variations. These findings provide a scientific basis for germplasm identification, conservation, and parental selection in C. eburneum Lindl. breeding. Full article

The effective control of foodborne salmonellosis relies on the rapid and reliable detection and identification of the pathogen. Reliable detection tools for identifying the most common Salmonella serovars should translate to a considerable alleviation of the health burden attributed to Salmonella. We have developed a PCR assay for the rapid identification of colonies of Salmonella enterica serovar Thompson, a common serovar. Genomic analyses of publicly available sequences of Salmonella Thompson revealed the presence of a unique, Thompson-specific fragment, which we have used to design a pair of oligonucleotides, STho-F and STho-R, for the PCR amplification of an 808 bp DNA fragment. Using crude DNA extracts, the 808 bp fragment was detected in 77 out of 78 isolates of S. Thompson (sensitivity = 98.7% n = 78 isolates) but not in any of the non-Salmonella organisms tested (n = 100; 100% specificity) nor in non-Thompson Salmonella serovars (n = 100; 100% specificity). The sensitivity (inclusivity) and specificity (exclusivity) indices of the PCR assay for S. Thompson met the standard regulatory requirements. The Thompson primer pair was compatible with other primers pairs in a multiplex PCR designed for three other common Salmonella serovars. Colonies belonging to the Enteritidis serovar (n = 100), Heidelberg serovar (n = 100), Typhimurium serovar (n = 100), and Thompson serovar (n = 77) were correctly designated, indicating excellent inclusivity and exclusivity scores for all four Salmonella serovars tested in a single multiplex PCR. Full article

Bacillus and Paenibacillus species are common and widely distributed microorganisms in food systems, often implicated in food spoilage and quality issues. Bacillus subtilis, in particular, has been associated with gas production and package bulging in seasoned foods. In this study, we developed a rapid and visual detection method for Bacillus subtilis by integrating (Recombinase Polymerase Amplification) RPA with (Clustered Regularly Interspaced Short Palindromic Repeats) CRISPR/Cas12a technology (designated as RPA-CRISPR/Cas12a). Specific RPA primers and probes were designed based on the conserved gyrB gene of Bacillus subtilis. Two sets of crRNA were designed according to the number of T-rich PAM sites on the RPA-amplified target sequence, and the reaction conditions were optimized in combination with the CRISPR/Cas12a trans-cleavage detection technology. Under optimized conditions, the crRNA3 guide (with a TT-rich PAM site) demonstrated superior cleavage efficiency compared to crRNA2 (TTT-rich PAM), while crRNA1 (TTTT-rich PAM) showed no activity. The assay achieved a detection limit of 150 pg/μL for genomic DNA and 5.5 CFU/mL for bacterial suspensions within 10 min at 37 °C. The method exhibited high specificity and sensitivity, providing a robust tool for early and on-site detection of Bacillus subtilis in food products. Full article

Background/Objectives: With the acceleration of global industrialization and continuous population growth, the world is increasingly confronted with the dual challenges of food insecurity and cultivated land contamination. The screening and breeding of rice varieties with superior agronomic traits and low heavy metal accumulation have therefore become important strategies for ensuring food safety and sustainable agricultural production. Methods: In this study, rice varieties carrying the Gn1a-i gene and exhibiting specific cadmium (Cd) accumulation characteristics were screened using a combination of molecular marker detection and cadmium accumulation evaluation. Specific loop-mediated isothermal amplification (LAMP) primers targeting the Gn1a-i gene were designed and combined with a lateral flow dipstick (LFD) assay to enable rapid genetic screening of rice varieties. A six-day hydroponic experiment under cadmium stress was conducted across three temperature ranges (15–20 °C, 22–27 °C, and 30–35 °C), and cadmium accumulation in different plant organs (roots, stem sheath, and leaves) was analyzed. Results: Seven varieties carrying the Gn1a-i gene, including Xiangwanxian 12, were identified among ten tested rice varieties. Xiangwanxian 12 was subsequently selected for further evaluation, with the high-cadmium-accumulating variety Yuzhenxiang used as a control. At 144 h, the total Cd content in the measured organs of Xiangwanxian 12 was 9.6%, 4.0%, and 23.2% lower than that of Yuzhenxiang under low, medium, and high temperatures, respectively (one-tailed t-test, p < 0.01 for all three temperatures). Conclusions: The integration of LAMP-based genotyping and physiological evaluation provides a novel and reliable strategy for identifying low-Cd rice germplasm. Xiangwanxian 12, which carries the Gn1a-i allele and exhibits consistently lower Cd accumulation than Yuzhenxiang, suggests potential as a candidate for breeding high-yield, low-Cd rice cultivars. Full article

Background: Papillary thyroid cancer (PTC) is the most common form of thyroid malignancy, with an incidence that has been steadily rising globally. Early and accurate diagnosis remains crucial for effective treatment and improved outcomes. MicroRNAs (miRNAs), small non-coding RNA molecules that regulate gene expression, have emerged as promising biomarkers in cancer research due to their stability and accessibility in serum. In this pilot study we compared the expression of 84 consistently reported, malignancy-associated serum miRNAs in patients with PTC (PTC group) and benign thyroid disease (Control group) as potential PTC markers. Methods: A focused panel containing primer assays for 84 human miRNAs that are consistently reported in the literature as being detectable and differentially expressed in serum in various organ-specific cancers was used to measure miRNA levels in serum samples from PTC (n = 8) and benign thyroid disease (n = 6) patients prior to thyroidectomy. Results: Among the 84 miRNAs analyzed, a panel of ten miRNAs showed numerical trends of differential expression between the two groups, including three upregulated (hsa-miR-150-5p, hsa-miR-21-5p, hsa-miR-23a-3p) and seven downregulated miRNAs (hsa-miR-17-5p, hsa-miR-17-3p, hsa-miR-200c-3p, hsa-miR-296-5p, hsa-miR-574-3p, hsa-miR-885-5p, hsa-miR-130-3p). The serum expression levels of hsa-miR-23a-3p were markedly elevated in patients with malignant nodules compared with those with benign lesions, while hsa-miR-574-3p was significantly downregulated in the PTC group. Conclusions: These findings warrant further investigation of hsa-miR-23a-3p and hsa-miR-574-3p in larger cohorts of patients with PTC to validate their potential clinical relevance. Full article

Marek’s disease (MD), caused by pathogenic Marek’s disease virus serotype 1 (MDV-1), is one of the most important avian immunosuppressive and neoplastic diseases and has led to huge economic losses to the poultry industry worldwide. Rapid and accurate clinical diagnosis is of great significance for efficient control of the disease. Herein, we have established a multiplex PCR (mPCR) method to simply differentiate all of the three types of MDV, using five specific primers targeting to MDV-1 oncogene meq or MDV-2 and MDV-3/HVT gB genes. Simultaneously, it can detect any type of virulent or vaccine MDV strains in one PCR reaction, with amplicons of the short (S) and long (L)-meq of MDV-1 strains, and the gB of MDV-2 and HVT vaccine strains. Non-specific amplifications of avian leukosis virus (ALV), reticuloendotheliosis virus (REV), or fowl adenovirus virus 4 (FAdV-4) were not observed, indicating a good specificity of this method. A total of 522 clinical samples of tumor-bearing or suspected diseased birds collected from 30 poultry farms were detected. The results demonstrated that the newly developed mPCR method accurately detected and differentiated epidemic MDV-1 infections and vaccine strains, and provided nearly 100% consistency for detecting clinical wild-type infections compared with conventional PCR amplification of the meq gene. Collectively, our data has provided a highly efficient method for early differential diagnosis of MD clinical cases, virus identification and future evaluation of vaccination efficacy in healthy chicken flocks, which would be meaningful for efficient control of the disease. Full article

C. pseudotuberculosis is a Gram-positive pathogenic bacterium that infects various animals, causing diseases such as caseous lymphadenitis, leading to significant economic losses in the livestock industry and posing zoonotic risks. This study targeted the conserved gyrA gene fragment of this bacterium, designed specific primers, optimized the reaction system and conditions, and established a high-resolution melting curve (HRM) detection method with potential utility for preliminary molecular screening. Validation showed that this method exhibits strong specificity, producing specific amplification only for the target biovars. The melting temperatures (Tm values) for the two biovars were 86.16 ± 0.05 °C and 86.92 ± 0.05 °C, respectively, allowing clear differentiation. It demonstrated high sensitivity, with minimum detection limits of 28 copies/μL and 25 copies/μL for standard plasmids of the ovis and equi biovars, respectively. The method also showed good reproducibility, with intra- and inter-batch coefficients of variation both below 1.0%. Applied to 133 clinical nasal swab samples from goats in Fujian Province, the method detected a positivity rate of 19.5% and indicates that a biovar equi-like gene fragment was detected in goat nasal swabs from Fujian Province via molecular screening. The HRM method developed in this study is sensitive, specific, simple, and cost-effective, enabling rapid detection and biovar differentiation of C. pseudotuberculosis. It is suitable for large-scale clinical sample screening and provides an efficient technical approach for epidemiological monitoring and precise control of the disease. Full article

Wheat, soybean, and peanut are recognized as major food allergens, with their prevalence rising globally, necessitating rapid and reliable detection methods. A new detection approach was developed in this research, which integrates Ladder-shape Melting Temperature Isothermal Amplification (LMTIA) with Proofreading Enzyme-Mediated Probe Cleavage (Proofman) technology to enable the concurrent identification of wheat, soybean, and peanut allergens. Compared with the loop-mediated isothermal amplification (LAMP) method under the experimental conditions set in this study, this approach can reduce the false-positive results associated with LAMP, and it does not rely on sophisticated instrumentation required by technologies like mass spectrometry. The GAG56D (wheat), Ara h 2.01 (peanut), and Lectin (soybean) genes were selected as target genes for the three allergens. Specific primers and probes were designed according to these target genes, and the reaction system was optimized. A systematic evaluation of the triplex Proofman-LMTIA method was then conducted regarding its specificity, sensitivity, limit of detection, and repeatability. Finally, the method’s practical applicability was validated using commercial products. The optimized system achieved simultaneous detection within 40 min at 61 °C, showing no cross-reactivity with common foods. The method demonstrated good sensitivity, with a sensitivity of 5 pg/μL for genomic DNA and a detection limit of 5% (w/w) in a powder matrix, along with excellent repeatability. In practical sample testing, the results were fully consistent with product label declarations, accurately identifying single and multiple allergen contaminations. The Proofman-LMTIA detection method, with its rapid, simple, sensitive, and specific characteristics, demonstrates significant potential for applications in food safety supervision. Full article

Reliable identification of seafood species is critical for fisheries management and product authentication, especially when morphological characteristics are lost during processing. In this study, a multiplex PCR system was developed to distinguish seven cuttlefish species (six Sepia spp. and Sepiella inermis) commercially distributed in the Korean seafood market. Species identity was first confirmed by amplifying a mitochondrial cytochrome c oxidase subunit I (COI) fragment (~658 bp) using universal primers (LCO1490/HCO2198), showing 99–100% sequence similarity to corresponding GenBank reference sequences. Analysis of genetic variation based on a 530 bp aligned region demonstrated complete interspecific differentiation without shared haplotypes among species. The number of haplotypes per species ranged from 5 to 21, with haplotype diversity values between 0.667 and 1.000. An extended COI fragment (~1200 bp) was further analyzed to identify diagnostic interspecific variation for marker development. Seven diagnostic single-nucleotide polymorphism (SNP) sites were identified and used to design species-specific forward primers with diagnostic nucleotides positioned at the 3′ termini. Distinct amplicons (220–1099 bp) were generated and clearly resolved by agarose gel electrophoresis. Because simultaneous amplification of all seven primer pairs reduced amplification efficiency, the assay was divided into two multiplex sets. Under optimized conditions (56 °C), each species produced a single expected band without cross-amplification. This multiplex PCR system provides a rapid and sequencing-free approach for reliable species discrimination and can be effectively applied to fisheries monitoring and seafood authentication in commercial supply chains. Full article

The genus Listeria comprises diverse bacteria with significant public health relevance, particularly Listeria monocytogenes. A comparative genomic analysis of ten representative Listeria species was conducted using 33 high-quality genome assemblies to investigate core and accessory genome dynamics and identify candidate diagnostic loci. Pangenome reconstruction was performed using the Roary Integer Linear Programming Bacterial Annotation Pipeline (RIBAP) to classify core, soft-core, and accessory genes, while average nucleotide identity (ANI) analysis assessed genomic relatedness across thresholds of 60–95%. Functional annotation of core and species-specific genes was conducted using Genome Annotation and Information Analysis (GAIA). Core genes were highly conserved and associated with essential cellular functions, whereas the accessory genome contributed to species-level diversification and ecological adaptation. Candidate molecular markers were derived from accessory genes and evaluated based on presence/absence across genomes, retaining loci present in ≥80% of target strains and absent in non-target strains. Experimental validation of selected primers was performed using two L. monocytogenes reference strains (ATCC 19117 and ATCC BAA-679) with conventional PCR and gel electrophoresis to confirm expected amplicon sizes and specificity. These findings establish a genome-informed, specificity-driven framework for marker development and highlight the accessory genome as a valuable source of diagnostic loci, supporting accurate detection, epidemiological surveillance, and food safety monitoring. Full article

Understanding salmonid spawning dynamics is critical for conserving cold-water river ecosystems amid increasing human and climate pressures. This study developed and validated a species-specific eDNA (Salmo cf. trutta and Thymallus thymallus) and evaluated its performance for seasonal spawning activity monitoring using droplet digital PCR (ddPCR). Species-specific primers and probes targeting mitochondrial nd5 (S. trutta) and cytb (T. thymallus) genes were designed and optimized as a duplex assay. Performance assessments included in vitro validation, cross-amplification testing, and determining the LOB, LOD, and LOQ. Field validation over a year at two spawning sites in the Gradac River, Serbia, involved seasonal eDNA sampling, filtration, extraction, and ddPCR analysis. Fish community composition was also assessed with electrofishing and metabarcoding. The assay showed high specificity and sensitivity, with LODs of 0.14 cp/µL and LOQs of 0.99 and 1.25 cpµL for S. trutta and T. thymallus. S. trutta eDNA peaked in late autumn during spawning, while T. thymallus remained at or below detection limits, reflecting its lower abundance and different spawning season. Filter type affected filtration efficiency but not eDNA yield. These findings confirm ddPCR-based eDNA as a powerful, non-invasive tool for monitoring salmonid spawning and seasonal changes, supporting adaptive fisheries management and conservation amid environmental changes. Full article

Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article

Intergeneric hybridization of Vanda and Papilionanthe holds promise for pyramiding superior ornamental and stress-tolerant traits, though systematic studies on their hybrids remain scarce. Using Vanda lamellata var. Boxallii (♀), Papilionanda ‘Hetty Henderson’ (♂), and 72 progenies, we investigated parent–progeny relationships via iPBS markers, spectral phenomics, and morphology, alongside floral water balance and thermotolerance. Six iPBS primers amplified 90 bands (92.98% polymorphism), confirming high genetic diversity. Spectral reflectance (400–1000 nm) revealed organ-specific genetic differentiation. Clustering analyses consistently indicated that progenies were genetically and phenotypically closer to the female parent, with spectral/morphological patterns matching genetic groupings. Resistance evaluations showed progenies had significantly stronger floral water storage capacity than both parents, while the female parent excelled in water transport traits. Progenies developed thicker petal/sepal cuticles, though the male parent exhibited superior thermotolerance indices. This study clarifies the genetic regulation of stress resistance in these hybrids, providing critical support for precise early screening in orchid breeding. Full article

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants. Full article

Indian senna (Senna alexandrina Mill.), a perennial medicinal species belonging to the family Fabaceae, holds significant therapeutic and commercial importance owing to its rich content of sennosides and rhein derivatives, which confer well-established laxative properties. Its high market demand, however, renders the species vulnerable to deliberate or inadvertent adulteration. While previous investigations have utilized functional marker systems such as SCoT (Start Codon Targeted Polymorphism)- and CBDP (CAAT Box Derived Polymorphism)-derived SCAR (Sequence Characterised Amplified Region) markers for genetic characterization, the present study is the first to report the development of sequence-specific RAPD- and ISSR-based SCAR markers consolidated into a single-tube multiplex PCR assay. Genomic DNA isolated from young leaves of S. alexandrina and its commonly encountered adulterant species was amplified using RAPD primer OPI-02 and ISSR primer UBC-835. Polymorphic amplicons were cloned, sequenced, and employed for the design of SCAR primers, which were rigorously validated for specificity. Species-specific SCAR markers were successfully integrated into a single multiplex reaction, enabling precise and unequivocal identification of S. alexandrina, Cassia fistula and Senna sophera. The multiplex amplification profiles were entirely consistent with corresponding uniplex assays, endorsing the method’s robustness and reproducibility. This streamlined, one-tube multiplex SCAR-PCR system represents a significant advancement toward reliable, high-throughput molecular authentication of Indian senna and its closely related medicinal plant species (adulterants). Full article