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Schistosomiasis, caused by trematodes of genus Schistosoma, is among the most seriously neglected tropical diseases. Although rapid surveillance of risk areas for Schistosoma transmission is vital to control schistosomiasis, the habitat and infection status of this parasite are difficult to assess. Environmental DNA (eDNA) analysis, involving the detection of extra-organismal DNA in water samples, facilitates cost-efficient and sensitive biomonitoring of aquatic environments and is a promising tool to identify Schistosoma habitat and infection risk areas. However, in tropical wetlands, highly turbid water causes filter clogging, thereby decreasing the filtration volume and increasing the risk of false negatives. Therefore, in this study, we aimed to conduct laboratory experiments and field surveys in Lake Victoria, Mbita, to determine the appropriate filter pore size for S. mansoni eDNA collection in terms of particle size and filtration volume. In the laboratory experiment, aquarium water was sequentially filtered using different pore size filters. Targeting >3 µm size fraction was found to be sufficient to capture S. mansoni eDNA particles, regardless of their life cycle stage (egg, miracidia, and cercaria). In the field surveys, GF/D (2.7 µm nominal pore size) filter yielded 2.5-times the filtration volume obtained with a smaller pore size filter and pre-filtration methods under the same time constraints. Moreover, a site-occupancy model was applied to the field detection results to estimate S. mansoni eDNA occurrence and detection probabilities and assess the number of water samples and PCR replicates necessary for efficient eDNA detection. Overall, this study reveals an effective method for S. mansoni eDNA detection in turbid water, facilitating the rapid and sensitive monitoring of its distribution and cost-effective identification of schistosomiasis transmission risk areas.

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Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

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The Yangtze River Estuary (YRE) is one of the areas in China most severely affected by harmful algal blooms (HABs). This study explored the distributive patterns of HABs in the YRE and how they are influenced by the El Niño-Southern Oscillation (ENSO) and other environmental factors. Quantitative real-time PCR (qPCR) was employed to detect and quantify the four predominant HAB species in the YRE, Karenia mikimotoi, Margalefidinium polykrikoides, Prorocentrum donghaiense, and Heterosigma akashiwo. Additionally, the study analyzed how turbidity, pH, salinity, and temperature influence these algae. Distribution of the four HAB species in the YRE area shows clear geographical variations: K. mikimotoi is predominantly found in the northwest and central sea areas, M. polykrikoides (East Asian Ribotype, EAR) is mainly distributed in the southeastern part, P. donghaiense is abundant in the northern regions, and H. akashiwo is especially prevalent at stations S26 and S27 in the northeastern part of the study area. HABs dominated by H. akashiwo and P. donghaiense were observed in the northeastern sea area of the YRE on July 22, 2020. Our study reveals that K. mikimotoi, M. polykrikoides (EAR), and P. donghaiense are mainly affected by turbidity, pH, and salinity, while temperature predominantly influences the blooms of H. akashiwo. Moreover, runoff in the YRE has a certain correlation with ENSO events, which may also impact the nutrient content of the region. The findings of this study illustrate the distributive patterns of the four HAB species under various ecological conditions in the YRE and emphasize the importance of establishing practical cases for future warning systems. To better understand how climate change affects HABs, exploring the link between ENSO and HABs is essential.

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Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

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Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

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Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

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Grapevine varieties from "Douro Superior" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer's stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed.

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Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/µL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

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In this study, we adopt an interdisciplinary approach, integrating agronomic field experiments with soil chemistry, molecular biology techniques, and statistics to investigate the impact of organic residue amendments, such as vinasse (a by-product of sugarcane ethanol production), on soil microbiome and greenhouse gas (GHG) production. The research investigates the effects of distinct disturbances, including organic residue application alone or combined with inorganic N fertilizer on the environment. The methods assess soil microbiome dynamics (composition and function), GHG emissions, and plant productivity. Detailed steps for field experimental setup, soil sampling, soil chemical analyses, determination of bacterial and fungal community diversity, quantification of genes related to nitrification and denitrification pathways, measurement and analysis of gas fluxes (N2O, CH4, and CO2), and determination of plant productivity are provided. The outcomes of the methods are detailed in our publications (Lourenço et al., 2018a; Lourenço et al., 2018b; Lourenço et al., 2019; Lourenço et al., 2020). Additionally, the statistical methods and scripts used for analyzing large datasets are outlined. The aim is to assist researchers by addressing common challenges in large-scale field experiments, offering practical recommendations to avoid common pitfalls, and proposing potential analyses, thereby encouraging collaboration among diverse research groups.•Interdisciplinary methods and scientific questions allow for exploring broader interconnected environmental problems.•The proposed method can serve as a model and protocol for evaluating the impact of soil amendments on soil microbiome, GHG emissions, and plant productivity, promoting more sustainable management practices.•Time-series data can offer detailed insights into specific ecosystems, particularly concerning soil microbiota (taxonomy and functions).

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Arbuscular mycorrhizal (AM) fungi are supposedly competing with ammonia-oxidizing microorganisms (AO) for soil nitrogen in form of ammonium. Despite a few studies directly addressing AM fungal and AO interactions, mostly in artificial cultivation substrates, it is not yet clear whether AM fungi can effectively suppress AO in field soils containing complex indigenous microbiomes. To fill this knowledge gap, we conducted compartmentalized pot experiments using four pairs of cropland and grassland soils with varying physicochemical properties. To exclude the interference of roots, a fine nylon mesh was used to separate the rhizosphere and mesh bags, with the latter being filled with unsterile field soils. Inoculation of plants with AM fungus Rhizophagus irregularis LPA9 suppressed AO bacteria (AOB) but not archaea (AOA) in the soils, indicating how soil nitrification could be suppressed by AM fungal presence/activity. In addition, in rhizosphere filled with artificial substrate, AM inoculation did suppress both AOB and AOA, implying more complex interactions between roots, AO, and AM fungi. Besides, we also observed that indigenous AM fungi contained in the field soils eventually did colonize the roots of plants behind the root barrier, and that the extent of such colonization was higher if the soil has previously been taken from cropland than from grassland. Despite this, the effect of experimental AM fungal inoculation on suppression of indigenous AOB in the unsterile field soils did not vanish. It seems that studying processes at a finer temporal scale, using larger buffer zones between rhizosphere and mesh bags, and/or detailed characterization of indigenous AM fungal and AO communities would be needed to uncover further details of the biotic interactions between the AM fungi and indigenous soil AO.

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Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (ß) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

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Vitellogenin (Vg) is a female-specific egg-yolk precursor protein, synthesized in the liver of fish in response to estrogens. In the present study, complete gene of phosvitinless vitellogenin (vgc) was sequenced, its 3D structure was predicted and validated by web-based softwares. The complete nucleotide sequence of vgc was 4126 bp which encodes for 1272 amino acids and showed the presence of three conserved domains viz. LPD_N, DUF1943 and DUF1944. The retrieved amino acid sequence of VgC protein was subjected to in silico analysis for understanding the structural and functional properties of protein. mRNA levels of multiple vg genes have also been quantified during annual reproductive cycle employing qPCR. A correlation has been observed between seasonal changes in gonadosomatic index with estradiol levels and hepatic expression of three types of vg genes (vga, vgb, vgc) during ovarian cycle of murrel. During preparatory phase, when photoperiod and temperature are low; low titre of E2 in blood induces expression of vgc gene. A rapid increase in the levels of E2 favours induction of vgb and vga genes in liver of murrel during early pre-spawning phase when photoperiod is long and temperature is high in nature. These results suggest that among three vitellogenin proteins, VgC is synthesized earlier than VgA and VgB during oogenesis.

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N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

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BACKGROUND: Abnormally expressed circular RNAs (circRNAs) are associated with many diseases and have important biological effects on the regulation of gene expression. However, the circRNA expression profile in incomplete radiofrequency ablation (RFA)-treated liver cancer (LC) patients has not been characterized. This study investigated the potential biological effects of differentially expressed (DE) circRNAs in an incomplete RFA-treated transplantation tumor model of human LC. MATERIAL/METHODS: A circRNA microarray was utilized to analyze changes in the circRNA expression profiles. CircRNA host gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were also conducted using computational biology. Quantitative real-time PCR (qPCR) was also performed on the selected DE-circRNAs to verify the reliability of the microarray. The circRNA/miRNA interactions were predicted by Arraystar software and confirmed by a dual-luciferase assay. RESULTS: Following RFA incomplete ablation, 76 DE-circRNAs were detected (|fold change |>1.5, P-value < 0.05), 21 of which were upregulated and 55 of which were downregulated. Computational biological analysis revealed that the T-cell receptor signaling pathway was the most significantly enriched pathway of the genes related to altered expression, as indicated by enrichment of LCK, AKT3 and DLG1. PCR results for the upregulated hsa_circRNA_103595 and downregulated hsa_circRNA_001264 indicated that the circRNA microarray sequencing results were reliable. Double luciferase reporter assays confirmed that hsa-miR-185-3p was the target miRNA of hsa_circRNA_103595. CONCLUSIONS: The current study confirmed the changes in the expression profiles of circRNAs in tumor transplantation models after incomplete ablation, these changes may play a crucial role in the pathophysiological process of residual cancer transplantation tumors. These findings could lead to new directions for investigating the molecular biological mechanisms underlying RFA-treated LC as well as new ideas for treating LC by regulating circRNAs.

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BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

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【Objective】 To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). 【Methods】 Based on the polymorphism of high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. 【Results】 The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR but positive by PCR-SSP, another sample was positive for 2DS1 by Q-PCR but negative by PCR-SSP. The two doubtful samples were genotyped by sequencing-based typing (PCR-SBT) for 2DS5 and 2DS1, respectively. PCR-SBT results confirmed that the results of Q-PCR test was correct. 【Conclusion】 The KIR Q-PCR method established in this paper can provide accurate and reliable results for testing the presence or absence of KIR genes.

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Candidatus Liberibacter spp is the most prevalent microorganism in the citrus plant, associated with Citrus Huanglongbing (HLB), which is transmitted by the psyllid vector. In Colombia, the vector Diaphorina citri Kugayama has been reported in different regions, but "Ca. Liberibacter asiaticus" (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1-OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a - CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we employed droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the ß-subunit of ribonucleotide reductase (RNR) gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.

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According to The Organization for Economic Co-operation and Development (OECD), demand for poultry meat and eggs consumption is growing consistently since poultry meat and eggs are readily available and cheap source for nutritional protein. As such, there is pressing demand from industry for improved protocols to determine chicken sex, especially in layer industry since only females can lay eggs. Extensive efforts are being dedicated to avoiding male chicks culling by developing in-ovo sexing detection methods. Any established in-ovo detection method will need to be validated by embryo genotyping. Therefore, there is a growing demand for fast, inexpensive, and precise method for proper discrimination between males and females in the poultry science community. Our aim with this study was to develop an accurate, high-throughput protocol for sex determination using small volumes of blood. We designed primers targeting the Hint-W gene within the W chromosome clearly distinguishing between males and females. In the interest of establishing an efficient protocol without the need for gel electrophoresis, crude DNA extraction without further purification was coupled with qPCR. We validated the accuracy of our method using established protocols and gonad phenotyping and tested our protocol with four different chicken breeds, day-nine embryos, day-old chicks and adult chicken. In summary, we developed a fast, cost-effective, and accurate method for the genotyping of sex chromosomes in chicken.

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The polymeric chain reaction (PCR) has come under fire for being time-consuming, requiring expensive equipments, and requiring the extraction and purification of nucleic acids. Here, an ultra-fast and sensitive detection platform without nucleic acid extraction solved the above problems. Firstly, the RoomTemp Sample Lysis Kit released the nucleic acid in 3 min and removed the inhibition to facilitate the amplification reaction. What's more, ultra-fast PCR (UF-PCR) can complete 40 cycles in just 15 min and 50 s. To improve the sensitivity and provide more convenient reading modes, CRISPR/Cas12a was mediated to detect Lumpy skin disease virus (LSDV). The platform output fluorescence and Lateral flow dipstick (LFD) signals. The actual detection limit was 2 × 101 copies·µL-1. The portable platform realized visualization, excellent sensitivity and quick speed. In summary, the field-friendly testing platform had great potential in practical testing.

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Neospora caninum is an obligate intracellular parasite that causes reproductive disorders and major economic losses in cattle, and induces neuromuscular disorders in canids. Exogenous infections are becoming increasingly important due to disease outbreaks. The sylvatic life cycle of N. caninum interferes with the domestic dog-ruminant life cycle, but understanding of it is scarce. The population of wild canids may play an important role in parasite dispersion. Feces from 42 grey wolves (Canis lupus) and 39 golden jackals (Canis aureus) were analyzed for the N. caninum Nc5 gene using a novel real-time PCR (qPCR) with a detection limit of 5 targets/µL in clinical samples. Three wolves (3/42; 7.1%) and one golden jackal (1/39; 2.6%) tested positive, which is the first detection of N. caninum in the population of grey wolves in Slovenia and the first detection of N. caninum DNA in the feces of a golden jackal. In addition to the grey wolf, we propose the golden jackal as a potential definitive host with hypothetical epidemiological importance for the sylvatic-domestic life cycle of N. caninum, due to its proximity to human habitats and its rapid expansion throughout Europe.