Multiplex PCR is one of the many variants routinely performed by polymerase chain reaction (PCR). Its advantage is especially rapid detection of a large number of products in one reaction using multiple primer sets. Optimization of the multiplex PCR protocol is important also for genes of metallothionein isoforms, so that we can carry out the amplification of more than one DNA sequence. Necessary part of this proces is the adjustments in temperature during the reaction steps and also intervention with the changes in the concentration of individual reagents and different combinations or the amount of each primer.
Cíl studie: Rychlá detekce nejčastějších aneuploidií pomocí metody multiplex QF PCR na nekultivovaných vzorcích choriové tkáně. Shrnutí výsledků aplikace multiplex QF PCR v managmentu péče o těhotné ženy v prvním trimestru gravidity. Typ studie: Původní práce. Název a sídlo pracoviště: Ústav lékařské genetiky a fetální medicíny FN a LF UP, Olomouc. Metodika: Vzorky choriové tkáně byly získány od 101 gravidních žen. Nekultivované vzorky byly zpracovány metodou multiplex QF PCR. Analyzovány byly STR lokusy chromozomů 13, 18, 21 a X, Y. Tyto markery byly amplifikovány ve 2 oddělených multiplex PCR reakcích za stejných podmínek a podrobeny fragmentační analýze v kapilární elektroforéze. Výsledky: Všech 101 analyzovaných vzorků choriové tkáně bylo úspěšně amplifikováno. V tomto souboru bylo metodou multiplex QF PCR celkem detekováno 16 patologií u plodů. Ve dvou případech se jednalo o triploidii, v sedmi případech byla zachycena trizomie chromozomu 21 – Downův syndrom, v šesti případech pak trizomie chromozomu 18 – Edwardsův syndrom a jednou byla odhalena monozomie gonozomu X – Turnerův syndrom. Závěr: Metoda multiplex QF PCR je nedílnou součástí screeningu prvního trimestru a poskytuje rychle dostupný a spolehlivý výsledek u vyšetřovaných pacientek.
Objective: Rapid detection of most frequent aneuploidies by the multiplex QF PCR method in non-cultured samples of chorial tissue. Summarized results of QF PCR method applied in the management of care of pregnant women in the first trimester of pregnancy. Type of study: An original contribution. Setting: Institute of Medical Genetics and Fetal Medicine, Faculty Hospital and Medical Faculty, Palacky University Olomouc. Methods: The samples of chorial tissue were obtained from 101 pregnant women. Non-cultured samples were processed by the multiplex QF PCR method. STR loci of chromosomes 13, 18, 21 and X and Y were analyzed. These markers were amplified in two separate multiplex PCR reactions under the same conditions and subjected to fragmentation analysis in capillary electrophoresis. Results: All 101 analyzed samples of chorial tissue were successfully amplified. In this group, 16 pathologies of the fetuses were detected by the multiplex QF PCR method. Triploidy was detected in two cases, trisomy of chromosome 21 – Down syndrome was found in seven cases, and trisomy of chromosome 18 – Edwards syndrome was found in six cases and monosomy of gonosome X – the Turner’ s syndrome was revealed once. Conclusions: The multiplex QF PCR method is an indispensable part of the screening of the first trimester and provides a rapidly available and reliable result in the examined patients.
- MeSH
- Aneuploidy MeSH
- Cytogenetic Analysis methods trends utilization MeSH
- Research Support as Topic MeSH
- Genetic Testing ethics methods MeSH
- Humans MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Prenatal Diagnosis methods trends utilization MeSH
- Pregnancy Trimester, First genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
The authors presei.t the case of non-HIV Kaposi's sarcoma treated successfully with Vinblastin (10 mg/m2 at two-week intervals to a total dose of 35 mg/m ), subsequently with interferon α-2b (5 million units s.c. three times per week for 16 weeks). After two years remission a complete relapse of KS occurred. Complete remission occurred again after repeated interferon α-2b treatment 5 million units three times per week for eight weeks, raised to 10 million units three times per week, repeatedly reduced to 5 million units three times per week up to a total 6-month period of administration. From the diagnostic aspect the authors draw attention to the importance of immunohistochemical examination using detection of CD34 antigen on endothelial and perivascular KS cells. In the discussion the authors draw attention to the virus etiology of KS HIV-positive and non-HIV (HHV 8), which explains the successful treatment with interferon α-2b in these conditions.
- MeSH
- Adult MeSH
- Drug Therapy methods MeSH
- Immunohistochemistry methods MeSH
- Interferon-alpha administration & dosage therapeutic use MeSH
- Sarcoma, Kaposi classification pathology therapy MeSH
- Humans MeSH
- Recurrence pathology MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Case Reports 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
One step multiple test line lateral flow immunoassay was designed in a competitive format for the detection of drugs of abuse (namely morphine, 3,4-methylen-dioxymethamphetamine, phenycyclidine and amphetamine) from oral fluids. For the labeling of antibodies, gold nanoparticles produced by the Turkevich method were used. It was found that saliva contains mucin glycoproteins which cause non-specific interactions on test lines. These undesirable interactions can be influenced by higher pH, higher buffer capacity, and a choice of suitable detergent for the sample pad buffer in combination with suitable sample pad material. Optimized sample pad buffer contained 100 mM Tris and 0.5% sodium deoxycholate to yield pH 9.0. Screening of all the materials (mainly sample pad, conjugate pad) was performed and Fusion 5 was selected in order to develop rapid test able to provide results within 10 minutes. The other key advantage of this format of the test, as compared to other immunoassays, is its low cost and simplicity requiring no sample or reagent preparation and no photodetector for its measurement.
- MeSH
- Chromatography, Affinity MeSH
- Immunochemistry MeSH
- Deoxycholic Acid MeSH
- Humans MeSH
- Mucins chemistry MeSH
- Nanoparticles MeSH
- Specimen Handling MeSH
- Substance Abuse Detection methods utilization MeSH
- Psychotropic Drugs analysis MeSH
- Saliva MeSH
- Gold MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.
- MeSH
- Escherichia coli O157 classification genetics MeSH
- Nucleic Acid Hybridization MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Listeria monocytogenes classification genetics MeSH
- Multiplex Polymerase Chain Reaction MeSH
- Salmonella classification genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Ciele: Analýza prenatálnych vzoriek za obdobie 2015–2020. Porovnanie miery detekcie klinicky relevantných variant cytogenetickou analýzou karyotypu a cytogenomickými metódami MLPA (Multiplex Ligation-Depent Probe Amplification) a mikročipmi (CMA – chromosomal microarray). Súbor a metodika: Analyzovaných bolo 1 029 prenatálnych vzoriek cytogenetickým hodnotením karyotypu (n = 1 029), cytogenomickými metódami MLPA (n = 144) a CMA (n = 111). Všetky nebalansované zmeny boli potvrdené metódou MLPA alebo CMA. Výsledky: Z analyzovaného súboru plodov, po odčítaní aneuploidií – 107 (10,40 %, n = 1 029), bolo analýzou karyotypu zachytených 22 štruktúrnych aberácií (2,39 %, n = 922) – deväť nebalansovaných zmien (0,98 %), 10 balansovaných zmien (1,08 %), jeden prípad nejasnej mozaiky (0,09 %), jeden prípad prítomnosti marker chromozómu (0,09 %) a jeden prípad diskordancie pohlavia (0,09 %). U 255 vzoriek s fyziologickým karyotypom indikovaných k cytogenomickému vyšetreniu bolo zachytených celkom osem (7,21 %, n = 111) patologických variant metódou CMA. Metódou MLPA bolo z týchto ôsmich patogénnych variant zachytených päť (3,47 %, n = 144). Celkový záchyt patogénnych variant metódami MLPA a CMA vrátane konfirmačných vyšetrení patologického karyotypu je 14 (5,14 %) a 17 (6,25 %) (n = 272). Záchyt patologických variant v skupine s izolovanými poruchami bol nižší než v skupine s mnohopočetnými poruchami (5,08 vs. 21,42 %). Záver: Potvrdila sa vyššia úspešnosť záchytu patologických variant so zmenou v počte kópií, metódou CMA než MLPA.
Objective: Analysis of prenatal samples from 2015 to 2020. Comparison detection rates of clinically relevant variants by cytogenetic karyotype analysis and cytogenomic MLPA (Multiplex Ligation-Depent Probe Amplification) and microarray methods (CMA – chromosomal microarray). Material and method: 1,029 prenatal samples were analyzed by cytogenetic karyotyping (N = 1,029), cytogenomic methods – MLPA (N = 144) and CMA (N = 111). All unbalanced changes were confirmed by MLPA or CMA. Results: From the analyzed set of fetuses, after subtraction of aneuploidies – 107 (10.40%, N = 1,029), 22 structural aberrations (2.39%, N = 922) – nine unbalanced changes (0.98%), 10 balanced changes (1.08%), one case of unclear mosaicism (0.09%), one case of presence of a marker chromosome (0.09%) and one case of sex discordance (0.09%) – were detected by karyotype analysis. A total of eight (7.21%, N = 111) pathological variants were detected by CMA in 255 samples with physiological karyotype indicated for cytogenomic examination. Five (3.47%, N = 144) of eight pathogenic variants were detected by MLPA method. The total capture of pathogenic variants by MLPA and CMA methods was 14 (5.14%) and 17 (6.25%) (N = 272), including confirmatory pathological karyotype testing. Detection of pathological variants in the isolated disorders group was lower than in the multiple disorders group (5.08 vs. 21.42%). Conclusion: A higher success rate for the detection of pathological copy number variation variants by the microarray method than by the MLPA method was confirmed.
- MeSH
- Clinical Studies as Topic MeSH
- Humans MeSH
- Microarray Analysis methods MeSH
- Mosaicism MeSH
- Multiplex Polymerase Chain Reaction methods MeSH
- Fetus MeSH
- Prenatal Diagnosis * MeSH
- Pregnancy MeSH
- DNA Copy Number Variations MeSH
- Congenital Abnormalities * diagnosis genetics MeSH
- Check Tag
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
