Cíl studie: Analýza klinického přínosu array vyšetření choriové biopsie (CVS) a návrh efektivnějšího postupu genetického vyšetření v I. trimestru. Typ studie: Retrospektivní studie. Název a sídlo pracoviště: Gennet, Centrum lékařské genetiky a reprodukční medicíny, Praha. Materiál a metodika: V rámci prenatální diagnostiky v I. trimestru bylo u 913 vzorků CVS provedeno QF-PCR (screening aneuploidií chromozomů 13,18, 21, X, Y) a stanovení karyotypu. Paralelně s těmito metodami bylo u 179 vzorků s normálním výsledkem z obou metod provedeno vyšetření SNP-array (Illumina HumanCytoSNP12 v2.1). Výsledky: Metodou QF-PCR bylo zachyceno 229 chromozomálních aneuploidií z 911 úspěšně provedených vyšetření (25 %). Konvenčními cytogenetickými metodami byly zachyceny nebalancované chromozomální aberace u 239 z 897 úspěšně vyšetřených plodů (27 %), v 95 % šlo o potvrzení výsledku QF-PCR (227/239), 10 nebalancovaných chromozomálních aberací nezahrnovalo chromozomy sledované metodou QF-PCR. Metodou array bylo u plodů s normálním výsledkem z obou výše uvedených metod odhaleno dalších 13 klinicky relevantních chromozomálních aberací (7,5 %). Závěr: Na základě analýzy našich dat a publikovaných studií jsme v laboratořích Gennetu navrhli nový algoritmus pro vyšetření choriových klků v I. trimestru. Hlavní změnou je nahrazení karyotypu metodou array u všech plodů, kde je normální výsledek z QF-PCR. Výsledkem bude efektivnější záchyt patologických klinicky relevantních chromozomálních aberací u vyšetřovaných plodů.

Objective: Array technology in chorionic villus sampling (CVS) – analysis of clinical benefit and a proposal of a more effective 1st trimester genetic testing policy. Design: Retrospective study. Setting: Gennet, Center of Medical Genetics and Reproductive Medicine, Prague. Material and methods: Total of 913 CVS were performed at Gennet between 2010–2014. All 913 samples were tested by QF-PCR rapid test for aneuploidy of chromosomes 13, 18, 21, X and Y and karyotyping following standard long term culture. Microarray analysis (Illumina HumanCytoSNP12 v2.1) was performed on 179 samples with normal result from both – QF-PCR and karyotyping. Results: At 229 samples the common chromosomal aneuploidy was detected using rapid QF-PCR (25% from 911 successful rapid tests). Conventional karyotyping revealed 239 unbalanced chromosome aberrations (27% from 897 successful cultivations). 227/239 (95%) positive karyotypes confirmed QF-PCR finding of common aneuploidies. 10 unbalanced chromosome aberrations were not covered by rapid QF-PCR test. Microarray analysis of samples with normal result from both– QF-PCR and karyotyping– revealed 13 clinically relevant chromosome aberrations (7.5%). Conclusion: New policy for chorionic villi testing at Gennet was established. Based on evaluation of the results of karyotyping, array and QF-PCR and analysis of published data we decided to replace karyotyping by microarray analysis in all cases of foetuses with normal results from QF-PCR. More effective detection of pathological and clinically relevant chromosome aberrations in examined foetuses is expected.

• Keywords
• QF-PCR, kvantitativní fluorescenční PCR,
• MeSH
• Algorithms MeSH
• Aneuploidy MeSH
• Chromosome Disorders * diagnosis   genetics   MeSH
• Cytogenetic Analysis methods   statistics & numerical data   MeSH
• Polymorphism, Single Nucleotide MeSH
• Karyotyping MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Chorionic Villi Sampling * MeSH
• Polymerase Chain Reaction MeSH
• Prenatal Diagnosis MeSH
• Pregnancy Trimester, First MeSH
• Retrospective Studies MeSH
• Oligonucleotide Array Sequence Analysis * MeSH
• Comparative Genomic Hybridization MeSH
• Pregnancy MeSH
• Ultrasonography, Prenatal MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Comparative Study MeSH

• MeSH
• Polymerase Chain Reaction methods   trends   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biologie

• MeSH
• Gene Amplification MeSH

• Conspectus
• Obecná genetika. Obecná cytogenetika. Evoluce
• NML Fields
• genetika, lékařská genetika
• biologie

Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.

• MeSH
• DNA, Viral genetics   MeSH
• Genome, Viral MeSH
• Multilocus Sequence Typing MeSH
• Polymerase Chain Reaction methods   MeSH
• Prophages classification   genetics   MeSH
• Siphoviridae classification   genetics   MeSH
• Comparative Genomic Hybridization MeSH
• Staphylococcus aureus genetics   virology   MeSH
• Viral Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

γδ T cells are intensively studied because their function in infection, allergy, autoimmune disease, cancer and post-transplant period is not yet fully understood. PCR-based techniques were established to study the γ variable (Vγ) and δ variable (Vδ) gene families. PCR product evaluation is routinely carried out by Southern blot analysis or the third complementarity-determining region spectratyping, but a fast and simple assessment of Vγ and Vδ gene family expression is missing. The aim of our study was to test capillary electrophoresis as a potential method for evaluating the composition of the γδ T-cell population. This report provides optimized PCR conditions for γδ T-cell receptor amplification. Further, it describes the utilization of capillary electrophoresis in the Agilent 2100 Bioanalyzer to evaluate the relative expression of Vγ and Vδ gene families after their amplification. An application of the methodology to peripheral blood mononuclear cell samples from patients during haemato-oncological treatment is shown. The described methodology is fast and simple to operate and is therefore suitable as a first screening of the γδ T-cell population composition in tissues of interest.

• MeSH
• Time Factors MeSH
• Adult MeSH
• Electrophoresis, Capillary methods   MeSH
• Hematologic Neoplasms genetics   pathology   MeSH
• Leukocytes, Mononuclear cytology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Multigene Family * MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   MeSH
• Receptors, Antigen, T-Cell, gamma-delta genetics   metabolism   MeSH
• Oligonucleotide Array Sequence Analysis methods   MeSH
• Case-Control Studies MeSH
• T-Lymphocytes cytology   metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc. However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.

• MeSH
• Apoptosis MeSH
• Fusion Proteins, bcr-abl metabolism   MeSH
• Drug Resistance, Neoplasm MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   genetics   MeSH
• Financing, Organized MeSH
• Humans MeSH
• RNA, Small Interfering pharmacology   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Biomarkers, Tumor genetics   MeSH
• Tumor Cells, Cultured MeSH
• Piperazines therapeutic use   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Cell Proliferation MeSH
• Proliferating Cell Nuclear Antigen genetics   MeSH
• Pyrimidines therapeutic use   MeSH
• Gene Expression Regulation, Leukemic MeSH
• RNA, Neoplasm genetics   metabolism   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Gene Expression Profiling MeSH
• Gene Silencing MeSH
• Check Tag
• Humans MeSH

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

• MeSH
• Aspergillus classification   genetics   isolation & purification   MeSH
• Molecular Diagnostic Techniques methods   MeSH
• Invasive Pulmonary Aspergillosis diagnosis   MeSH
• Humans MeSH
• Polymerase Chain Reaction methods   MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.

• MeSH
• Biosensing Techniques instrumentation   MeSH
• Deoxyadenine Nucleotides chemistry   MeSH
• Equipment Design MeSH
• DNA analysis   MeSH
• Electrochemical Techniques instrumentation   MeSH
• Limit of Detection MeSH
• Metallocenes chemistry   MeSH
• Microelectrodes MeSH
• Seawater analysis   MeSH
• Oxidation-Reduction MeSH
• Polymerase Chain Reaction instrumentation   MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH

Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.

• MeSH
• Yersinia Infections diagnosis   microbiology   MeSH
• Humans MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Foodborne Diseases diagnosis   microbiology   parasitology   MeSH
• Toxoplasma genetics   isolation & purification   MeSH
• Toxoplasmosis diagnosis   parasitology   MeSH
• Yersinia enterocolitica genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Long non-coding RNAs (lncRNAs) are defined as RNA molecules longer than 200 nucleotides with poor protein-coding capacity and key functions in regulation of gene expression. Dysregulations of lncRNAs (e.g. HOTAIR and MALAT I) were detected in plasma of breast cancer (BC) patients. Plasma samples are examined as liquid biopsies for purposes of non-invasive diagnostics therefore the research of plasma lncRNAs as potential plasma biomarkers became highly topical. 84 lncRNAs were profiled in 18 plasma samples - 9 BC patients and 9 age-matched healthy - using Human Inflammatory Response & Autoimmunity RT2 lncRNA PCR Array. Total RNA from plasma samples was isolated using miRNeasy Serum/Plasma Kit. Although a pre-amplification recommended for quantification from small starting RNA amounts was used, only 3 lncRNAs (A2ML1-AS1, GAS5 and SNHG5) were detected in all plasma samples. A total of 72 lncRNAs (e.g. HOTAIR or MALAT I) were detected only in some samples and 9 lncRNAs were not detected in any samples. No significant differences were observed in levels of plasma lncRNAs between the BC patients and healthy controls despite the fact that our panel contained also the lncRNAs whose levels were previously reported as significantly different in plasma or cancer tissues (e.g. GAS5, HOTAIR, MALAT I) in BC patients. Detection of lncRNAs in plasma is due to their low concentrations quite difficult as compared with tissues. Our findings suggest that analysis of plasma lncRNAs using this technology is not suitable for use as non-invasive diagnostic tool in BC patients.

• MeSH
• Humans MeSH
• Breast Neoplasms diagnosis   genetics   MeSH
• Polymerase Chain Reaction * MeSH
• Gene Expression Regulation, Neoplastic MeSH
• RNA, Long Noncoding blood   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH