Search Results (134)

Accurate normalization is crucial for reliable gene expression quantification and depends on stably expressed housekeeping genes (HKGs) as internal controls. However, HKGs expression varies with developmental stage, tissue type, and treatments, potentially introducing bias and compromising data accuracy. Thus, validating candidate reference genes under defined conditions is essential. Reynoutria, also known as giant Asian knotweeds, is a Polygonaceae family genus of several medicinal plants producing a diverse array of specialized metabolites of pharmacological interest. Outside their native range, these plants are also noxious invasive weeds, causing significant environmental and economic threats. Research on stable reference genes in these species is limited, with a primary focus on R. japonica. To enable accurate gene expression analysis related to specialized metabolism and natural product biosynthesis, we aimed to identify the most stable reference genes across the most common species: R. japonica Houtt., R. sachalinensis (F. Schmidt) Nakai, and their hybrid—R. × bohemica Chrtek & Chrtková. In this study, we evaluated twelve candidate HKGs (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, eIF6A, SKD1, YLS8, and NDUFA13) across three tissue types (rhizomes, leaves, and flowers) from three Reynoutria species sampled at peak flowering. Primer specificity and amplification efficiency were confirmed through standard-curve analysis. We assessed expression stability using ΔCt, geNorm, NormFinder, and BestKeeper, and generated comprehensive rankings with RefFinder. Our integrated analysis revealed organ- and species-dependent stability differences, yet identified up to three reference genes suitable for interspecific normalization in Reynoutria. This represents the first systematic, comparative validation of HKGs across closely related knotweed species, providing a robust foundation for future transcriptomic and functional studies of their specialized metabolism and other biological processes. Full article

Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 are four major foodborne pathogenic bacteria found in meat and meat products, which pose significant threats to human health. In this study, we developed specific loop-mediated isothermal amplification (LAMP) primers targeting these four pathogenic bacteria. Following the optimization of system components and reaction parameters, four rapid and simplified LAMP-based detection assays were established, which enabled the visual detection of these four pathogenic bacteria within 40–50 min. The three established LAMP assays targeting L. monocytogenes, S. aureus, and E. coli O157:H7 achieved species-level discrimination, whereas the LAMP method for Salmonella exhibited genus-level specificity. The detection limits of the LAMP assays were determined as follows: 1.8 × 10¹ colony forming units (CFU)/mL for L. monocytogenes, 5.1 × 10¹ CFU/mL for S. aureus, 1.2 × 10¹ CFU/mL for S. enterica, and 3.3 × 10³ CFU/mL for E. coli O157:H7, with sensitivity improved by 10–1000-fold compared to conventional PCR. The developed LAMP assays were used to analyze 52 meat and meat product samples, and 7 samples were positive, which was consistent with the results of the conventional PCR and culture-based methods, demonstrating an accuracy rate of 100% for the LAMP methods. In conclusion, the established LAMP assays exhibit high specificity, enhanced sensitivity, and result visualization, making them suitable for on-site rapid detection in food safety monitoring. Full article

Tusavirus 1 of species Protoparvovirus incertum 1 (family Parvoviridae) was first identified in humans and later in small ruminants (caprine and ovine). This study reports the full-length coding sequences (~4400–4600 nt) of three novel tusavirus-related protoparvoviruses from ovine (“misavirus”, PV540792), for the first time bovine (“sisavirus”, PV540793) and subsequently from caprine (“gisavirus” PV540850/51) fecal samples, using next-generation sequencing (NGS) and PCR techniques. Their NS1, VP1 and VP2 proteins shared 61–63% amino acid identities with each other and with tusaviruses, suggesting these three viruses belong to three novel species in the genus Protoparvovirus. Phylogenetic analyses placed them with tusaviruses on a separate main branch, implying a shared origin among these most likely ruminant protoparvoviruses. A small-scale epidemiological investigation on 318 ruminant enteric samples using novel generic NS1 primers found misavirus in 14/51 (27.5%) ovine and sisavirus in 19/203 (9.4%) bovine samples from multiple Hungarian farms. Tusavirus was present in 5/51 (9.8%) ovine and 15/62 (24.2%) caprine samples, all from one farm. The highest prevalences for all three viruses were found in animals aged 2–12 months, though sporadic cases were also found in other age groups. Partial NS and VP sequence-based phylogenetic trees showed virus-specific lineages for misa-, sisa-, gisa- and tusaviruses, with various strains forming sub-lineages. These findings suggest the presence of multiple genotypes and/or members of additional species, which was supported by a VP sequence-based hierarchical cluster analysis. The study’s viruses were mostly phylogenetically separated by host; however, two bovine sisavirus strains with diverse phylogenetic localizations in the NS (belonging to bovine sisaviruses) and VP1 trees (distantly related to ovine misaviruses) could indicate previous (interspecies?) recombination events. Full article

Gastrointestinal nematode infections are due to a wide number of helminthic genera and species, representing a major concern in goat and sheep farming and leading to different health issues and a general economic loss. Traditional diagnostic tools do not allow for a specific identification and, although a shift towards molecular diagnostic techniques is ongoing, species or genus-specific diagnosis is still poorly implemented. This study describes the development of a novel real-time PCR method for diagnosing Haemonchus sp. and its relative abundance in mixed infections in grazing ruminants. The method employs two primer/probe sets targeting the 18S-rRNA-ITS1-5.8S-ITS2 region: one shared by all strongylids (GEN) and another specific to Haemonchus sp. (HAEM). The method demonstrated optimal efficiency and determination coefficients when applied to serial dilutions of DNA extract. It was then applied in Faecal Egg Count Reduction Test (FECRT) trials conducted on five sheep and five goat farms in northeastern Italy. Seven farms were suspected of overall resistance and only one farm of Heamonchus-related resistance. The results proved the genus-specific approach as valuable in interpreting treatment outcomes and showing concerning levels of anthelmintic treatment ineffectiveness. Further research and sensitization activity is required to encourage the adoption of the method by local farmers and veterinarians. Full article

Two main adenoviral diseases have been described in pigeons: pigeon adenovirus type 1 (PiAdV-1) and pigeon adenovirus type 2 (PiAdV-2), which belong to the genus Aviadenovirus under the family Adenoviridae. PiAdV-1 and PiAdV-2 are highly pathogenic to pigeons, leading to considerable losses worldwide. To date, there is little information on the epidemiological distribution of PiAdV-1 and PiAdV-2 in pigeons due to the lack of detection and differentiation platforms for these two viruses. High-resolution melting technology (HRM) has been widely used for developing detection and differentiation platforms, with the melting profile based on the GC content in the real-time PCR (qPCR-HRM) system. This study designed and synthesized a pair of specific primers on the basis of the characteristic variations of the 52K genes of PiAdV-1 and PiAdV-2, then the detection and differentiation qPCR-HRM platform was established after conditional optimization. The results showed that this method had good specificity; it could only specifically detect PiAdV-1 and PiAdV-2, with no cross-reaction with other pigeon-origin pathogens that occur in pigeons. This method had high sensitivity, with the lowest detection limits at 57 copies/µL (for PiAdV-1) and 56 copies/µL (for PiAdV-2). This method had good intra-group and inter-group coefficients of variation, both of which were less than 1.5%. Field samples for the epidemiological surveillance and investigation data of PiAdV-1 and PiAdV-2 were checked. We found only PiAdV-2-positive samples in meat pigeons, but the percentages of PiAdV-1-positive, PiAdV-2-positive, and coinfection-positive samples among the racing pigeons were 5.71%, 14.29%, and 2.86%, respectively. To our knowledge, this is the first report for the simultaneous detection and differentiation of PiAdV-1 and PiAdV-2 using the qPCR-HRM platform. Our study also provided evidence of PiAdV-1 and PiAdV-2 coinfection in racing pigeons, but further studies are needed. Full article

The proliferation of harmful cyanobacteria, particularly Microcystis, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic Microcystis in freshwater environments. We targeted the secA gene, specific to the Microcystis genus, and the mcyA gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected Microcystis at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring Microcystis dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms. Full article

As a perennial medicinal plant in the Zingiber genus, Amomum villosum Lour. faces agricultural challenges due to its prolonged vegetative and reproductive growth phases, which hinder efficient pollination and delay fruiting. To address this limitation, the present study aimed to identify the FD gene involved in regulating flowering in A. villosum to provide a basis for research on the molecular mechanisms of early fruiting cultivars. Based on the differentially expressed gene AvFD1 obtained from the transcriptome database of early fruiting plants and controls, specific primers were designed for PCR to clone the full-length sequence of AvFD1. The characteristics of the cloned AvFD1 gene were analyzed using online bioinformatics software. The expression profiles of AvFD1 in various tissues and in 1- and 2-year bearing A. villosum varieties were investigated by quantitative real-time PCR. This study successfully cloned the FD1 gene sequence of A. villosum, marking the first reported characterization of this gene in the species. Tissue-specific expression analysis revealed significantly elevated AvFD1 expression levels in stolon tips and flower buds compared to tender leaves, suggesting its potential role as a positive regulator of flowering initiation. The obtained sequence establishes essential molecular data for subsequent functional validation of AvFD1 in A. villosum. Full article

Understanding the dominant populations and biological functions of endophytic nitrogen-fixing bacteria in apple plants is of great significance for the healthy growth management and sustainable development of apple cultivation. In this study, we investigated the community diversity and potential plant growth-promoting abilities of endophytic nitrogen-fixing bacteria in different tissues of apple trees by combining high-throughput sequencing of the nifH gene with traditional isolation and cultivation techniques. Sequencing results revealed that the endophytic bacteria were affiliated with 10 phyla, 14 classes, 30 orders, 42 families, and 72 genera. Rhizobium was the dominant genus in the roots and twigs, while Desulfovibrio dominated the leaf tissues. The diversity and richness of endophytic bacteria in the roots were significantly higher than those in the leaves. Using four types of nitrogen-free media, a total of 138 presumptive endophytic nitrogen-fixing bacterial strains were isolated from roots, leaves, and twigs. These isolates belonged to 32 taxonomic groups spanning 5 phyla, 8 classes, 11 orders, 13 families, and 18 genera. The nifH gene was successfully amplified from the representative strains of all 32 groups using specific primers. Nitrogenase activity among the isolates ranged from 26.86 to 982.28 nmol/(h·mL). Some strains also exhibited the ability to secrete indole-3-acetic acid (IAA), solubilize phosphate and potassium, and produce siderophores. Six individual strains and three microbial consortia were tested for their plant growth-promoting effects on apple tissue culture seedlings. All treatments showed growth-promoting effects to varying degrees, with the RD01+RC16 consortium showing the most significant results: plant height, number of leaves, and chlorophyll content were 2.4, 3.3, and 4.2 times higher than those of the control, respectively. These findings demonstrate the rich diversity of endophytic nitrogen-fixing bacteria in apple plants and their promising potential for application in promoting host plant growth. Full article

The Vibrio genus represents a critical group of bacterial pathogens in the marine environment globally, leading to massive mortality in the aquaculture industry. Diagnosing vibriosis, an infection caused by Vibrio species, in clinical samples poses challenges due to its non-specific clinical manifestations. In this study, we developed a TaqMan probe-based multiplex real-time PCR method for the simultaneous detection and quantification of four Vibrio pathogens: Vibrio anguillarum (Va), Vibrio alginolyticus (Val), Vibrio harveyi (Vh), and Vibrio scophthalmi (Vsc). The assay targets conserved intra-species regions and specific inter-species regions using specific primers and TaqMan probes to ensure specificity. Sensitivity analysis demonstrated that the multiplex real-time PCR assay could simultaneously detect the four different bacteria, with detection limits of 26–60 copies per reaction, making it 100 times more sensitive than conventional PCR assays. Additionally, the assay exhibited high reproducibility, with intra- and inter-group coefficients of variation below 1.4%. A total of 63 clinical samples was analyzed using this established assay, which successfully detected both single and mixed infections. These results demonstrate that the multiplex quantitative PCR assay is a rapid, specific, and sensitive diagnostic tool for the detection of Va, Val, Vh, and Vsc, making it suitable for monitoring these bacteria in both single- and co-infected clinical samples. Full article

Species of the genus Diaporthe have a mating-type system with the two mating types MAT1-1 and MAT1-2, like other ascomycetes. They can either be heterothallic, which means that any isolate only possesses one of the two mating types and needs a mating partner for sexual reproduction, or homothallic, which means that they possess both mating types and are self-fertile. For several Diaporthe species, no sexual reproduction has been observed so far. Using PCR with primers specific to the defining genes MAT1-1-1 and MAT1-2-1, we determined the mating types of 33 isolates of Diaporthe caulivora, D. eres, D. longicolla, and D. novem from central Europe. In addition, we partially sequenced the mating-type genes of 25 isolates. We found that different D. longicolla isolates either possess MAT1-1-1 or MAT1-2-1, making the species heterothallic, which is in contrast to previous studies and the general assumption that D. longicolla only reproduces asexually. D. eres and D. novem were also found to be heterothallic. Using genomic sequence information and re-sequencing of DNA and RNA, we identified the MAT1-1-1 gene in D. caulivora and present here the full sequence of the mating-type locus of this homothallic species. Finally, we used sequence information from MAT1-1-1 and MAT1-2-1, respectively, for improved phylogenetic resolution of our isolates. Full article

The Achromobacter genus comprises 22 species and various genogroups. Some species with higher virulence or antibiotic resistance are more likely to cause chronic infections in people with cystic fibrosis (CF). Current identification methods often fail to accurately distinguish between the species or result in misidentifications due to biochemical similarities. This study aims to develop an accurate qPCR protocol for species-level identification that is applicable in clinical diagnostic laboratories. Whole-genome sequencing of clinical isolates from different Achromobacter species identified species-specific single-nucleotide polymorphisms (SNPs) in two 16S gene regions. Based on these SNPs, two sets of primers and qPCR probes were designed to generate unique identification profiles. Thermal profiles were optimized, and qPCR was performed on serial bacterial DNA dilutions to determine the detection limit (LOD). Four probes successfully identified three species: A. xylosoxidans, A. dolens, and A. insuavis. Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg/µL to 1 pg/µL. Combined probes achieved 100% sensitivity, with specificity ranging from 97.95% to 100%. This qPCR protocol enables accurate species identification, overcoming the limitations of current methods, and represents a reliable tool for clinical diagnostics. Full article

Lettuce (Lactuca sativa L.) plants showing leaf curl and vein enation symptoms were found in Yunnan province, China. PCR detection with genus-specific primers revealed that symptomatic lettuce plants were infected with Begomovirus. The full-length viral component and satellite molecules were obtained by RCA, restriction enzyme digestion, PCR, cloning and DNA sequencing. A viral component (YN-2023-WJ) and three satellite molecules (YN-2023-WJ-alpha1, YN-2023-WJ-alpha2 and YN-2023-WJ-beta) were obtained from diseased lettuce plants. YN-2023-WJ exhibited the highest nt identity at 97.1% with pepper leaf curl Yunnan virus isolated from cigar plants. YN-2023-WJ-beta displayed the highest nt identity at 93.9% with tomato leaf curl China betasatellite. YN-2023-WJ-alpha1 showed the highest nt identity at 94.7% with ageratum yellow vein alphasatellite. YN-2023-WJ-alpha2 shared the highest nt identity at 75.6% with gossypium mustelinum symptomless alphasatellite and vernonia yellow vein Fujian alphasatellite. Based on the threshold for the classification of Begomovirus, Betasatellite and Alphasatellite, YN-2023-WJ was designated as a new isolate of PepLCYnV, YN-2023-WJ-beta as a new isolate of ToLCCNB and YN-2023-WJ-alpha1 as a new member of AYVA, whereas YN-2023-WJ-alpha2 was identified as a new geminialphasatellite species, for which the name pepper leaf curl Yunnan alphasatellite (PepLCYnA) is proposed. To the best of our knowledge, this is the first report of L. sativa L. infection by PepLCYnV associated with ToLCCNB, AYVA and PepLCYnA, and L. sativa L. is a new host plant of Begomovirus. Full article

Fowl adenovirus (FAdV) belongs to the Aviadenovirus genus within the Adenoviridae family. FAdVs are widely distributed and associated with various diseases in poultry, including adenoviral gizzard erosion (AGE), hepatitis-hydropericardium syndrome (HHS), and inclusion body hepatitis (IBH). In this study, we developed a multiplex conventional PCR for simultaneously detecting FAdV-4, -8b, and -11 by targeting the hexon gene. The multiplex PCR was optimized for primer concentrations and thermocycling conditions. The optimal primer concentration combination was set at 0.125 μM for FAdV-4, -8b, and 0.25 μM for FAdV-11. Under these conditions, the limit of detection (LOD) was 10³ copies/μL of plasmid standards for FAdV-4, -8b, and -11. These results demonstrated that the developed multiplex PCR method exhibits high specificity and sensitivity, with no observed cross-reactivity among these serotypes or with other poultry viruses. Therefore, this multiplex PCR will be an effective tool for accurate serotyping of FAdV-4, -8b, and -11, enabling more precise identification and differentiation of these three serotypes. Full article

Rape (Brassica napus) is an important oilseed crop widely cultivated worldwide. Due to its relatively short evolutionary and domestication history, its intra-species genetic diversity is limited. Radish (Raphanus sativus), belonging to a different genus but the same family as B. nupus, possesses an abundance of excellent gene resources. It is commonly used for B. nupus germplasm improvement and genetic basis expansion, making it one of the most important close relatives for distant hybridization. In the present study, a novel method for detecting alien chromosome fragments, called Anchor Mapping of Alien Chromosome (AMAC) was used to identify radish chromosome segments in the progeny of rape–radish interspecific hybrids. Based on the AMAC method, 126,861 pairs of IP (Intron Polymorphism) and 76,764 pairs of SSR (Simple Sequence Repeat) primers were developed using the radish Rs1.0 reference genome. A total of 44,176 markers (23,816 pairs of IP and 20,360 pairs of SSR markers) were predicted to be radish genome specific-single-locus (SSL) markers through electronic PCR analysis among four R. sativus, one B. napus, one B. rapa, one B. juncea, and one B. juncea reference genome. Among them, 626 randomly synthesized SSL markers (478 SSL IP markers and 148 SSL SSR markers) were used to amplify the genome of 24 radish samples (R. sativus), 18 rape (B. napus), 2 Chinese cabbage (B. rapa), 2 kale (B. oleracea), and 2 mustard (B. juncea) samples, respectively. Then, 333 SSL markers of the radish genome were identified, which only amplified in the radish genome and not in any Brassica species genome, including 192 IP markers and 141 SSR markers. Furthermore, these validated SSL markers were used to identify alien chromosome fragments in Ogura-CMS restorer line 16C, Ogura-CMS sterile line 81A, and their hybrid-Yunyouza15. In 16C, one marker, Rs1.0025823_intron_3, had an amplification product designated as anchor marker for the alien chromosome fragment of 16C. Afterwards, four novel radish genome-specific IP markers were found to be flanking the anchor marker, and it was determined that the alien chromosome segment in 16C originated from the region 8.4807–11.7798 Mb on radish chromosome R9, and it was approximately 3.2991 Mb in size. These results demonstrate that the AMAC method developed in this study is efficient, convenient, and cost-effective for identifying excellent alien chromosome fragments/genes in distant hybrid progeny, and it can be applied to the molecular marker-assisted breeding and hybrid identification of radish and Brassica crop species. Full article

Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica (“glass milk”) in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD₅₀ of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen’s kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis. Full article