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This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. Unlike other DNA-based assays, this method uses digoxigenin- and biotin-labeled primers. This eliminates the need for a separate step of hybridization of the PCR product with labeled probes. The PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. Primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, +/-25 cycles were optimal. The sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites. DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. This method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers.
- MeSH
- DNA Primers genetics MeSH
- Enzyme-Linked Immunosorbent Assay methods MeSH
- Leishmania major genetics isolation & purification MeSH
- Leishmania genetics isolation & purification MeSH
- Leishmaniasis diagnosis parasitology MeSH
- Mice MeSH
- Polymerase Chain Reaction methods MeSH
- DNA, Protozoan analysis genetics MeSH
- Base Sequence MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Quantification of monoclonal immunoglobulin free light chains (FLCs) in serum is used increasingly in clinical practice for the diagnosis, prognostic assessment, and treatment monitoring of monoclonal gammopathies. It is used as an adjunct to standard serum protein electrophoresis and immunofixation. However, methods for FLC quantification need further standardization and validation. METHODS: The Czech Myeloma Group and the Czech Society of Clinical Biochemistry have initiated an interlaboratory study where six laboratories collaborating with the primary myeloma treatment centres measured FLC concentrations in 12 serum samples from patients with monoclonal gammopathies. RESULTS: Repeatability of the measurements in five laboratories was calculated based on differences between the results of duplicate measurements. We found that repeatability depended more on the laboratory than on the device used for measurement. CONCLUSIONS: The study revealed several weak points in the methodology, including the need for a uniform sample dilution procedure. Interlaboratory reproducibility was comparable with values achieved in the NEQAS programme. Because the κ/λ ratio cannot be measured with high precision, κ and λ FLC concentrations should be used where possible. Due to its impact on the clinical management of patients with gammopathy, FLC quantification needs to become a part of the regular quality control cycle in myeloma centres.
- MeSH
- Clinical Laboratory Techniques methods standards MeSH
- Immunoglobulin Light Chains analysis blood MeSH
- Middle Aged MeSH
- Humans MeSH
- Multiple Myeloma blood diagnosis MeSH
- Paraproteinemias blood diagnosis MeSH
- Reference Standards MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
The aims of our research involved to investigate DYS-14 copy number variations in healthy males, to quantify extracellular DNA in maternal circulation in normal versus complicated pregnancies, and to study variations in the DYS-14 copy number in extracellular male fetal DNA. Fifty-five healthy males, 43 uncomplicated male singleton pregnancies (23 sampled at the 16th week and 20 sampled at the 36th week), and 15 pregnancies with placental insufficiency (PI)-related complications (mean 34.1 weeks) were analyzed using real-time PCR with DYS-14 sequence, sex determining region Y (SRY), and beta-globin (GLO) genes used as markers. Increased levels of extracellular DNA were detected in PI-related complications relative to gestational age-matched controls (SRY, p < 0.001; DYS-14, p = 0.007; GLO, p < 0.001). When the mean + 2SD (standard deviation) of controls was used as a cutoff, SRY, DYS-14, and GLO achieved 91.7%, 68.8%, and 94.4% accuracy, respectively, for differentiation between normal and complicated pregnancies. Considerable variations in the DYS-14 copy number in healthy males (mean 52.6) and extracellular DNA were found. A lower DYS-14 copy number was observed in PI-related complications (mean 83.5) compared to uncomplicated pregnancies (16th week: mean 114.2, p = 0.02; 36th week: mean 142.8, p = 0.04). The DYS-14 copy number was higher in extracellular DNA throughout gestation relative to healthy males. We concluded that, regarding interindividual copy number variations, the DYS-14 sequence is not an optimal marker for extracellular fetal DNA quantification for differentiation between normal and complicated pregnancies.
- MeSH
- Child MeSH
- DNA blood MeSH
- Adult MeSH
- Gene Dosage MeSH
- Gestational Age MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Chromosomes, Human, Y genetics MeSH
- Mothers MeSH
- Adolescent MeSH
- Young Adult MeSH
- Placental Insufficiency diagnosis genetics MeSH
- Fetus MeSH
- Child, Preschool MeSH
- Prenatal Diagnosis MeSH
- Sex-Determining Region Y Protein genetics MeSH
- Cell Cycle Proteins MeSH
- Case-Control Studies MeSH
- Pregnancy MeSH
- Pregnancy Outcome MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
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
- Pest Control, Biological MeSH
- DNA, Helminth genetics MeSH
- Snails parasitology MeSH
- Nematoda genetics isolation & purification parasitology MeSH
- Host-Parasite Interactions MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Soil parasitology MeSH
- Rhabditoidea genetics isolation & purification parasitology MeSH
- DNA, Ribosomal genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Switzerland MeSH
Microarray studies are capable of providing data for temporal gene expression patterns of thousands of genes simultaneously, comprising rich but cryptic information about transcriptional control. However available methods are still not adequate in extraction of useful information about transcriptional regulation from these data. This study presents a dynamic model of gene expression which allows for identification of transcriptional regulators using time series of gene expression. The algorithm was applied for identification of transcriptional regulators controlling 40 cell cycle regulated genes of Saccharomyces cerevisiae. The presented algorithm uses a dynamic model of time continuous gene expression with the assumption that the target gene expression profile results from the action of the upstream regulator. The goal is to apply the model to putative regulators to estimate the transcription pattern of a target gene using a least squares minimization procedure. The procedure iteratively tests all possible transcription factors and selects those that best approximate the target gene expression profile. Results were compared with independently published data and good agreement between the published and identified transcriptional regulators was found.
- MeSH
- Algorithms MeSH
- Genes, cdc MeSH
- Financing, Organized MeSH
- Transcription, Genetic MeSH
- Gene Regulatory Networks MeSH
- Linear Models MeSH
- Least-Squares Analysis MeSH
- Models, Genetic MeSH
- Gene Expression Regulation, Fungal MeSH
- Saccharomyces cerevisiae genetics MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Gene Expression Profiling MeSH
- Transcription Factors analysis MeSH
- Computational Biology MeSH
- Publication type
- Evaluation Study MeSH
- Comparative Study MeSH
BACKGROUND: Thermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR. RESULTS: Species specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low. CONCLUSIONS: Proposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.
- Publication type
- Journal Article MeSH
The development and validation of real-time quantitative PCR (qPCR) assays specific to all seven Eimeria species that cause coccidiosis in the chicken is described. The presented work utilizes previously published assays for Eimeria maxima, E. necatrix and E. tenella and adds assays for E. acervulina, E. brunetti, E. mitis and E. praecox. These assays target unique single copy sequences derived from sequence characterized amplified region (SCAR) markers. All seven qPCR markers were sequenced from multiple strains and confirmed to be non-polymorphic and identical to the original SCAR sequence. Sequences conserved within each species were chosen with the aim of developing genuinely universal markers, providing global coverage. An exact match for the primers and TaqMan(®) probe during PCR cycling enables precise relative quantification of multiple species in a mixture regardless of the strains present. All markers utilized in these qPCR assays are absolutely species-specific and support reproducible quantification across a wide linear range, unaffected by the presence of non-target species or other contaminating DNA. The sensitivity of these assays indicates that DNA equivalent to a single sporulated oocyst can be consistently detected. These assays will be a valuable tool from both industry and research perspectives. Comparison of our panel of qPCR assays with results derived by microscopy, the traditional Gold Standard, using poultry farm field samples support their efficacy.
- MeSH
- Species Specificity MeSH
- Eimeria classification isolation & purification MeSH
- Coccidiosis parasitology veterinary MeSH
- Chickens MeSH
- Poultry Diseases parasitology MeSH
- Polymerase Chain Reaction methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.
- MeSH
- Staining and Labeling methods MeSH
- Cell Adhesion MeSH
- Cell Line MeSH
- Cell Cycle MeSH
- Cytophotometry methods MeSH
- Diploidy * MeSH
- DNA analysis chemistry MeSH
- Sodium Dodecyl Sulfate chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- HeLa Cells MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Cell Count instrumentation methods MeSH
- DNA Replication * MeSH
- Reproducibility of Results MeSH
- Cell Survival MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
