real-time interactions
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BACKGROUND: Polymerase chain reaction (PCR) has become a common technique offering fast and sensitive analysis of DNA in food/feed samples. However, many substances, either already present in the sample or introduced during sample processing, inhibit PCR and thus underestimate the DNA content. It is therefore necessary to identify PCR inhibition in order to correctly evaluate the sample. RESULTS: We designed and validated a synthetic plasmid DNA that can be used to detect and quantify PCR inhibition. The DNA sequence, appropriate primers and probe, were designed in silico, synthesized and the sequence was inserted into a plasmid vector. The performance of the plasmid was verified via calibration curves and by performing the assay in the presence of various DNAs (crops, fungus, bacterium). The detection of PCR inhibition was assessed using six inhibiting substances with different modes of action, substances used in sample processing (EDTA, ethanol, NaCl, SDS) and food additives (sodium glutamate, tartrazine). The plasmid performance proved to be reproducible and there were no interactions with other DNAs. The plasmid was able to identify the presence of the inhibitors in a wide range of concentrations. CONCLUSION: The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.
Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.
- MeSH
- DNA fungální chemie genetika MeSH
- Glomeromycota genetika růst a vývoj MeSH
- kořeny rostlin mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- Medicago sativa mikrobiologie MeSH
- mikrobiální interakce MeSH
- mitochondriální DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- počet mikrobiálních kolonií metody MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.
- MeSH
- komplementární DNA genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- rostlinné geny * MeSH
- sekvenční analýza RNA MeSH
- Silene genetika MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Salmonella Enteritidis is the main serovar of poultry origin in humans, but its complex interaction with certain avian cells is still not fully understood. Previously we identified several genes significantly induced in chicken embryo fibroblasts (CEFs) by the wild-type strain S. Enteritidis 11 (SE 11). In the present study, we raised the question whether virulence-attenuated mutants of this strain would induce altered expression of the newly identified fibroblast genes associated with immune and non-immune functions of CEFs. Gene expression was evaluated by real-time PCR following challenge by the parental strain SE 11 and its virulence attenuated mutants lacking flagellin gene fliD only or fliD and the serovar-specific virulence plasmid pSEV. As a result, deletion mutants induced a lower expression of all immune genes, but an increased expression of the non-immune genes G0S2 and ENO2 relative to the parental strain. Our data indicate the importance of flagella and pSEV in modulation of virulence and host response in this model. We demonstrated, for the first time ever, an increased induction of survival genes G0S2 and ENO2 by virulence-attenuated mutants of S. Enteritidis.
- MeSH
- bakteriální proteiny genetika MeSH
- fibroblasty mikrobiologie MeSH
- flagella genetika MeSH
- interakce hostitele a patogenu MeSH
- kuřecí embryo MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- nemoci drůbeže imunologie mikrobiologie MeSH
- plazmidy genetika MeSH
- Salmonella enteritidis genetika patogenita MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.
Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.
- MeSH
- biologická kontrola škůdců MeSH
- DNA helmintů genetika MeSH
- hlemýždi parazitologie MeSH
- hlístice genetika izolace a purifikace parazitologie MeSH
- interakce hostitele a parazita MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- půda parazitologie MeSH
- Rhabditoidea genetika izolace a purifikace parazitologie MeSH
- ribozomální DNA genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Švýcarsko MeSH
Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.
- MeSH
- akutní myeloidní leukemie genetika MeSH
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- mladiství MeSH
- mladý dospělý MeSH
- nádorové buňky kultivované MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- protein - isoformy MeSH
- proteiny WT1 genetika MeSH
- senioři MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report a novel approach to biosensor-based observations of biomolecular interactions which enables real-time monitoring of biomolecular interactions in complex media. This approach is demonstrated by investigating the interaction between the human chorionic gonadotropin (hCG) and its antibody in blood plasma using a surface plasmon resonance biosensor and a dispersionless microfluidics system. The real-time binding data obtained in blood plasma are compared with those obtained in buffer and blood plasma using a conventional method. It is also demonstrated that the proposed approach can enhance the capability of the biosensor to detect biomolecules in complex samples in terms of detection time and sensitivity. In the model experiment, this approach is shown to enable direct detection of hCG in blood plasma at levels which are five times lower than those detected using the conventional detection approach.
- MeSH
- biosenzitivní techniky metody MeSH
- choriogonadotropin krev chemie MeSH
- lidé MeSH
- mikrofluidika MeSH
- povrchová plasmonová rezonance metody MeSH
- protilátky chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs. Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role. The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.
