•High-throughput qPCR-based chip was developed to simultaneously quantify 27 HPBs.•The HPB-Chip showed high specificity at the species level and high sensitivity via low LoQ.•The HPB-Chip was ...successfully applied to wastewater systems in urban environment.
Waterborne pathogens are threatening public health globally, but profiling multiple human pathogenic bacteria (HPBs) in various polluted environments is still a challenge due to the absence of rapid, high-throughput and accurate quantification tools. This work developed a novel chip, termed the HPB-Chip, based on high-throughput quantitative polymerase chain reactions (HT-qPCR). The HPB-Chip with 33-nL reaction volume could simultaneously complete 10,752 amplification reactions, quantifying 27 HPBs in up to 192 samples with two technical replicates (including those for generating standard curves). Specific positive bands of target genes across different species and single peak melting curves demonstrated high specificity of the HPB-Chip. The mixed plasmid serial dilution test validated its high sensitivity with the limit of quantification (LoD) of averaged 82 copies per reaction for 25 target genes. PCR amplification efficiencies and R2 coefficients of standard curves of the HPB-Chip averaged 101 % and 0.996, respectively. Moreover, a strong positive correlation (Pearson’ r: 0.961–0.994, P < 0.001) of HPB concentrations (log10 copies/L) between HPB-Chip and conventional qPCR demonstrated high accuracy of the HPB-Chip. Subsequently, the HPB-Chip has been successfully applied to absolutely quantify 27 HPBs in municipal and hospital wastewater treatment plants (WWTPs) after PMA treatment. A total of 17 HPBs were detected in the 6 full-scale WWTPs, with an additional 19 in the hospital WWTP. Remarkably, Acinetobacter baumannii, Legionella pneumophila, and Arcobacter butzler were present in the final effluent of each municipal WWTP. Overall, the HPB-Chip is an efficient and accurate high-throughput quantification tool to comprehensively and rapidly quantify 27 HPBs in the environment.
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The main ingredient in marzipan is sweet almonds (Prunus dulcis var. dulcis). Bitter almonds (Prunus dulcis var. amara) are also added to create a more intense flavour. In Germany the proportion of ...bitter almonds in marzipan is limited to a maximum of 12%. The use of debittered bitter almonds is prohibited. However, currently no analytical technique can reliably monitor compliance with this legal requirement. In this study we used next-generation sequencing technology to sequence the plastid genomes of six sweet cultivars (Larguetta, Ferragnes, Nonpareil, Carmel, Monterey, and Marcona) and six bitter almond batches of different origin (Iran, Morocco, Kyrgyzstan, Spain, Syria, and Turkey) to locate single nucleotide variants (SNVs). Sweet and bitter almonds share the same maternal lineage, but sequence variants on the plastid genome were present in most bitter almond populations. To exploit these differences for detection of bitter almonds in processed products, we used the double-mismatch allele-specific qPCR (DMAS-qPCR) for two polymorphic loci (rpoB, rps4). We provide evidence that the rps4 variant was detectable in almond raw pastes containing (debittered) bitter almonds, showing that the DNA sequence was sufficiently stable throughout food processing. A clear distinction from sweet almonds was observed for samples that contained at least 8% of bitter almonds. Our results present a promising approach to detect adulterations with bitter almonds in marzipan.
•Bitter almonds show variable plastid haplotypes.•The double-mismatch allele-specific qPCR (DMAS-qPCR) can detect polymorphic loci of rpoB and rps4 in bitter almonds.•Bitter almond admixtures in marzipan can be identified with DMAS-qPCR.
Quantification of functional genes involved in nitrogen (N) transformation improves our understanding of N-cycling microbial population responses to environmental disturbance. Agricultural N ...fertilization affects N-cycling gene abundances in soil, but the general patterns and variability of N cycling gene abundances in response to N fertilization have yet to be synthesized. We conducted a meta-analysis comprising 47 field studies in agricultural ecosystems. We included five marker genes important to N-cycling: nifH (encoding nitrogenase; key enzyme for N fixation), amoA (encoding ammonia monooxygenase; key enzyme for nitrification), nirK and nirS (encoding nitrite reductase; key enzyme for denitrification), and nosZ (encoding nitrous oxide reductase; key enzyme for denitrification). We found that N fertilization had no effect on the abundance of nifH, but significantly increased archaeal amoA (31%), bacterial amoA (313%), nirK (53%), nirS (40%) and nosZ (75%), respectively. N fertilizer form (inorganic versus organic) strongly affected the response of most selected N-cycling genes to N fertilization; organic fertilizers often had a much stronger effect than inorganic fertilizers. N fertilization duration, crop rotation, and soil pH were also important factors regulating the response of most N-cycling genes to N fertilization. Genes involved in nitrification and denitrification were significantly correlated with each other. Improvement in understanding of the response of N-cycling gene abundance to enhanced N input will help develop quantitative models of N availability and N fluxes and improve strategies for reducing reactive N gas emissions and N management in agricultural ecosystems.
•N fertilization had no effect on the abundance of gene involved in N-fixation.•N fertilization increased the abundance of genes in nitrification and denitrification.•N fertilization form and soil pH regulate N-cycling genes response to N fertilization.•Genes in nitrification and denitrification were significantly correlated with each other.
•A total of 51 known miRNAs and 95 novel miRNAs were identified from the nine small RNA libraries.•6 down-regulated DE miRNAs responded to the hypo-salinity stress.•1 up-regulated and 5 ...down-regulated DE miRNAs responded to the hyper-salinity stress.•931 and 768 potential target genes were respectively predicted from the hypo- and hyper-salinity stress.•Genes involved in the vesicle-mediated transport and metal ion binding responded to the hypo-salinity stress.•Genes related to the signal transduction and metabolic process responded to the hyper-salinity stress.
The Hong Kong oyster, Crassostrea hongkongensis, is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis.
Nickel compounds in dissolved form or as nanoparticles may affect planktonic invertebrates in marine ecosystems. Here, we assessed the physiological (naupliar mortality, egg production, egg hatching ...success) and molecular (quantitative gene expression) responses of the crustacean copepods Acartia clausi (indigenous Mediterranean species) and Acartia tonsa (model organism in ecotoxicology), to nickel nanoparticles (NiNPs) and nickel chloride (NiCl2), over time. We also measured NPs size and the temporal release of Ni ions in aqueous solution, through dynamic light scattering (DLS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively.
Nauplii of A. clausi were highly vulnerable to NiCl2 in the 48 h acute test, with an EC50 in the range of Ni concentrations measured in polluted waters. Females of both species exhibited a decreased egg production and hatching success after the 4-day exposure to NiNPs. Molecular responses in A. clausi incubated in NiNPs and NiCl2 showed a stronger up- or down-regulation, compared to A. tonsa, of genes associated with detoxification (phospholipid-hydroperoxide glutathione peroxidase, glutathione-S-transferase sigma), oxidative stress (superoxide dismutase), nervous system functioning (acetylcholinesterase), and oogenesis (vitellogenin).
In conclusion, new information was here obtained on the effects of different forms of nickel on physiological and molecular responses of A. clausi, that could help to identify biomarker genes of exposure to be used as early-warning indicators. Our results also highlighted the need of employing indigenous copepod species to better evaluate the ecotoxicological impact of pollutants in different geographical area.
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•Acartia clausi nauplii were highly sensitive to NiCl2.•Nickel nanoparticles decreased female egg production in both species.•Nanoparticles release Ni ions, decreasing copepod hatching success.•NiCl2 down-regulated oxidative stress and nervous system genes in A. clausi.
Background
Reliable biomarkers to differentiate gastrointestinal cancer (GIC) from chronic inflammatory enteropathy (CIE) in dogs are needed. Fecal and serum microRNAs (miRNAs) have been proposed as ...diagnostic and prognostic markers of GI disease in humans and dogs.
Hypothesis/Objectives
Dogs with GIC have fecal and serum miRNA profiles that differ from those of dogs with CIE. Aims: (a) identify miRNAs that differentiate GIC from CIE, (b) use high‐throughput reverse transcription quantitative real‐time PCR (RT‐qPCR) to establish fecal and serum miRNA panels to distinguish GIC from CIE in dogs.
Animals
Twenty‐four dogs with GIC, 10 dogs with CIE, and 10 healthy dogs, all client‐owned.
Methods
An international multicenter observational prospective case‐control study. Small RNA sequencing was used to identify fecal and serum miRNAs, and RT‐qPCR was used to establish fecal and serum miRNA panels with the potential to distinguish GIC from CIE.
Results
The best diagnostic performance for distinguishing GIC from CIE was fecal miR‐451 (AUC: 0.955, sensitivity: 86.4%, specificity: 100%), miR‐223 (AUC: 0.918, sensitivity: 90.9%, specificity: 80%), and miR‐27a (AUC: 0.868, sensitivity: 81.8%, specificity: 90%) and serum miR‐20b (AUC: 0.905, sensitivity: 90.5%, specificity: 90%), miR‐148a‐3p (AUC: 0.924, sensitivity: 85.7%, specificity: 90%), and miR‐652 (AUC: 0.943, sensitivity: 90.5%, specificity: 90%). Slightly improved diagnostic performance was achieved when combining fecal miR‐451 and miR‐223 (AUC: 0.973, sensitivity: 95.5%, specificity: 90%).
Conclusions and Clinical Importance
When used as part of a diagnostic RT‐qPCR panel, the abovementioned miRNAs have the potential to function as noninvasive biomarkers for the differentiation of GIC and CIE in dogs.
As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum , an ...apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase ( mdhfr-ts ) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum , a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains Nc ΔGRA7 and Nc ΔROP40 . For Nc ΔGRA7 , the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the Nc ΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.
The high-throughput antibiotic resistance gene (ARG) qPCR array, initially published in 2012, is increasingly used to quantify resistance and mobile determinants in environmental matrices. Continued ...utility of the array; however, necessitates improvements such as removing or redesigning questionable primer sets, updating targeted genes and coverage of available sequences. Towards this goal, a new primer design tool (EcoFunPrimer) was used to aid in identification of conserved regions of diverse genes. The total number of assays used for diverse genes was reduced from 91 old primer sets to 52 new primer sets, with only a 10% loss in sequence coverage. While the old and new array both contain 384 primer sets, a reduction in old primer sets permitted 147 additional ARGs and mobile genetic elements to be targeted. Results of validating the updated array with a mock community of strains resulted in over 98% of tested instances incurring true positive/negative calls. Common queries related to sensitivity, quantification and conventional data analysis (e.g. Ct cutoff value, and estimated genomic copies without standard curves) were also explored. A combined list of new and previously used primer sets is provided with a recommended set based on redesign of primer sets and results of validation.
MicroRNAs (miRNAs) play pivotal roles in gene regulation and their dysregulation is implicated in various diseases, including cancer. Current methods for miRNA analysis often involve complex ...procedures and high costs, limiting their clinical utility. Therefore, there is a critical need for the development of simpler and more cost-effective miRNA detection techniques to enable early disease diagnosis. In this study, we introduce a novel one-enzyme for miRNA one-step detection method using Taq DNA polymerase, termed OSMOS-qPCR. We optimized the PCR buffer, PCR program, Taq DNA Polymerase concentrations and reverse PCR primer concentrations, resulted in a wide linear range from 100 fM to 0.001 fM (R2 > 0.98 for each miRNA), the detection limit for OSMOS-qPCR was 0.0025 fM. Furthermore, OSMOS-qPCR demonstrates excellent specificity to differentiation of less than 0.1 % nonspecific signal. Finally, we demonstrated the robust amplification efficiency, enabling the detection of trace amounts of cell-free miRNA in serum samples, and the excellent discrimination ability between gastrointestinal cancers and control subjects (AUC value = 1.0) if combined two miRNAs. The development of OSMOS-qPCR offering a simpler, cost-effective, and efficient detection method, has the potential to be non-invasive strategy for early detection of gastrointestinal cancers.
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•First report the feasibility of single Taq DNA Polymerase for detection of miRNA.•One-step reverse transcriptase-free miRNA detection system with high specificity and low detection limit below 0.01 fM.•High accuracy for detecting cell-miRNA in serum for early detection of gastrointestinal cancers.
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