Polymerasová řetězová reakce (PCR) je metoda používaná pro namnožení specifického úseku DNA. Pro své vlastnosti se stala tato technika oblíbenou a v praxi hojně používanou metodou. V současné době rozlišujeme tři generace PCR – tradiční PCR (I. generace), kvantitativní PCR s fluorescenční detekcí v reálném čase – qPCR (II. generace) a digitální PCR – dPCR (III. generace). dPCR je robustní a citlivá metoda, díky čemuž nachází uplatnění v mnoha oblastech jako je mikrobiologie, analýza potravin, onkologie apod. Využívá se především pro absolutní kvantifikaci a/nebo detekci vzácných cílů. Její výhodou je, v porovnání s qPCR, přesnější kvantifikace nezávislá na počtu amplifikačních cyklů a kalibrační křivce.

Polymerase chain reaction (PCR) is a technique used to amplify a specific DNA segment. For its qualities, this technique has become popular and widely used method in practise. Nowaday, we distinguish three generations of PCR – traditional PCR (1st Generation), quantitative PCR with fluorescence detection – qPCR (2nd generation) and digital PCR – dPCR (3rd generation). dPCR is robust and sensitive method, which is applied in many fields such as microbiology, food analysis, oncology, etc. It is mainly used for absolute quantification and / or detection of rare targets. Its advantage, in comparison to qPCR, is more precise quantification independent of the number of amplification cycles and the calibration curve.

• Klíčová slova
• digitální PCR,
• MeSH
• DNA MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• polymerázová řetězová reakce klasifikace   metody   MeSH
• senzitivita a specificita MeSH
• techniky amplifikace nukleových kyselin MeSH
• Publikační typ
• práce podpořená grantem MeSH

Glutathione S-transferase M1 is a cytosolic enzyme important for the biotransformation of xenobiotics in various human tissues. The aim of this study was to perform genetic analysis of deletion polymorphism in the GSTM1 gene by using the technology of digital PCR. For genotyping, the QX100 Droplet Digital PCR System was used. The absolute quantity of GSTM1 copies was normalized to the ß-globin reference gene. In our experimental group, the prevalence of GSTM1*0 null variant was 67 %. Frequencies of GSTM1*1/*1 (n=5), GSTM1*0/*1 (n=23), and GSTM1*0/*0 (n=22) genotypes were 10 %, 46 %, and 44 %, respectively. Digital PCR seems to be an available and reliable technology for GSTM1 deletion polymorphism genotyping.

• Klíčová slova
• digitální PCR,
• MeSH
• delece genu MeSH
• diagnostické techniky molekulární metody   MeSH
• dospělí MeSH
• genetická predispozice k nemoci * MeSH
• genotypizační techniky * metody   přístrojové vybavení   MeSH
• klinická studie jako téma MeSH
• kvantitativní polymerázová řetězová reakce metody   přístrojové vybavení   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory diagnóza   genetika   MeSH
• počítače využití   MeSH
• polymerázová řetězová reakce * metody   přístrojové vybavení   MeSH
• polymorfismus genetický MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Východiská: Podstatou moderných postupov liečby onkologických pacientov je v dnešnej dobe zacielenie konkrétnych molekúl zapojených do bunkovej signalizácie asociovanej s nádorovou iniciáciou a progresiou. Úspech uvedeného prístupu závisí od správne zvoleného diagnostického testu s vysokou citlivosťou, ktorý identifikuje výskyt a hladinu vybraných biomarkerov u pacientov pre selekciu tých, ktorí budú na liečivo reagovať a budú z neho benefitovať. Vývoj nových technológií a modernizácia tých známych, prispievajú k inováciám molekulárnej charakterizácie karcinómov, ktorá umožňuje detekciu mutačného stavu pacienta s vysokou citlivosťou a špecifickosťou. Cieľ: V práci diskutujeme o využití polymerázovej reťazovej reakcie (PCR) tretej generácie, tzv. droplet digitálnej PCR (ddPCR), v molekulárnej diagnostike karcinómov. Podľa štúdií uvedených v našom prehľade predstavuje ddPCR sľubný nástroj pri vytváraní genetického profilu pacientov s onkologickým ochorením. Optimalizácia a presná validácia môžu preto umožniť postupnú implementáciu ddPCR do klinickej praxe v oblasti onkológie.

Background: Nowadays, modern treatment methods for cancer patients are based on targeting specific molecules involved in cellular signaling system associated with tumor initiation and progression. The success of such approach depends on a correctly chosen diagnostic test with high sensitivity that identifies the occurrence and level of biomarkers in patients to select those who will respond and benefit from the treatment. The development of new technologies and the upgrades of the known ones contribute to the innovations in molecular characterization of cancer, which allows the detection of patient’s mutational status with high sensitivity and specificity. Purpose: Here, we discuss the utilization of the third-generation type of polymerase chain reaction (PCR), droplet digital PCR (ddPCR), in the molecular dia­gnostics of oncology diseases. According to the studies reported in our review, ddPCR represents a promising tool in genetic profiling of cancer patients. Therefore, the optimization and precise validation may enable gradual implementation of ddPCR into clinical practice in the field of oncology.

• Klíčová slova
• ddPCR,
• MeSH
• diagnostické techniky molekulární metody   MeSH
• lidé MeSH
• nádorové biomarkery MeSH
• nádory * diagnóza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

PCR metoda se velmi krátce od svého objevení stala rutinní metodou molekulárně bio­logických výzkumných laboratoří a nepostradatelným nástrojem dia­gnostické medicíny. Za dobu svého využívání byla rozvinuta do řady variant, které specificky reagují na potřeby výzkumu a dia­gnostiky co do použitého vstupního materiálu a jeho množství, podmínek reakce a nově vyvinutých technologií. Předložená práce stručně shrnuje jednotlivé PCR přístupy s důrazem na jejich využití v onkologickém výzkumu a praxi.

Since its discovery, PCR has become a conventional method of molecular bio­logy research laboratories and an indispensable tool in dia­gnostic medicine. Multiple variants of the PCR technique were developed, which enable the analysis of different bio­logical materials at different amounts and reaction conditions. This article briefly summarizes the PCR approaches and points out their applications in oncological research and practice. Key words: polymerase chain reaction (PCR) – real‑time PCR – digital PCR – clinical oncology This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), BBMRI_CZ (LM2010004), GACR 13-00956S and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 10. 2. 2014 Accepted: 1. 4. 2014

• Klíčová slova
• digitální PCR,
• MeSH
• kvantitativní polymerázová řetězová reakce * metody   MeSH
• lékařská onkologie MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádory * diagnóza   genetika   MeSH
• polymerázová řetězová reakce * dějiny   metody   trendy   MeSH
• polymorfismus genetický genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Polymerase chain reaction (PCR) is one of the main techniques of molecular biology. Currently, there is a boom of digital PCR which allows precise, sensitive and reproducible quantification without using a calibration curve. Due to its features, digital PCR has a wide analyti-cal application in many areas (e.g. biomedical applications or food analysis). Presently, there are several types of digi-tal PCR that differ from each other particularly in the way of dispersing the sample into many aliquots of very small volumes. All platforms allow multiplex analyses based on the use of differently labelled probes and/or different con-centrations of probes labelled with one fluorescent color. This can reduce the financial and time requirements for a single sample analysis. The advantages and disadvantages of each type of digital PCR are summarized in this article.

Pleural and peritoneal infections cause substantial morbidity and mortality. Traditional diagnostic methods rely on the cultivation of clinical samples, which usually takes days to obtain report and holds a low detection sensitivity. In this study, we evaluated a 5-fluorescent-channel droplet digital PCR (ddPCR) system and 5 assay panels for culture-independent rapid pathogen detections directly from pleural and peritoneal fluid samples. Traditional culture of the same sample was used as reference. A total of 40 pleural fluid samples and 19 peritoneal fluid samples were tested in this study. Twenty-five positives including 4 polymicrobial infections by culture and 26 positives including 11 polymicrobial infections by ddPCR were detected for pleural fluid samples; 14 positives including 2 polymicrobial infections by culture and 15 positives including 3 polymicrobial infections by ddPCR were detected for peritoneal fluid samples. Klebsiella pneumoniae was the most common bacterium detected both in pleural and in peritoneal fluid samples. The sensitivity of the ddPCR assay for pleural and peritoneal fluid samples was 96% (95% confidence interval (CI) = 79.65 to 99.90%) and 92.86% (95% CI = 66.13 to 99.82%), respectively. The turnaround time of the ddPCR assay was approximately 3 h comparing with 38.30 ± 22.44 h for culture-based identifications. Our results demonstrated that the ddPCR assay is a rapid and sensitive method for identifying pathogens responsible for pleural and peritoneal infections and would be a promising approach for early diagnosis and optimizing treatment of infections.

• MeSH
• kvantitativní polymerázová řetězová reakce * MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).

• MeSH
• DNA krev   genetika   MeSH
• genotyp MeSH
• genotypizační techniky metody   MeSH
• gestační stáří MeSH
• krevní skupiny - systém Rh-Hr genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• odběr vzorku krve metody   MeSH
• plod metabolismus   MeSH
• polymerázová řetězová reakce metody   MeSH
• prenatální diagnóza metody   MeSH
• reprodukovatelnost výsledků MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

• MeSH
• aneuploidie MeSH
• Downův syndrom * diagnóza   genetika   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• prenatální diagnóza metody   MeSH
• těhotenství MeSH
• trizomie MeSH
• volné cirkulující nukleové kyseliny * genetika   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Goeckerman therapy (GT) represents an effective treatment of psoriasis including a combination of pharmaceutical grade crude coal tar (CCT) and ultraviolet irradiation (UV-R). Coal tar contains a mixture of polycyclic aromatic hydrocarbons. The best known carcinogenic polyaromate - benzo[a]pyrene is metabolized into a highly reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). Glutathione S-transferase M1 (GSTM1) catalyses the conjugation of drugs, toxins and products of oxidative stress with glutathione. The aim of the study is to found possible associations between GSTM1 genotypes and the level of BPDE-DNA adducts in 46 psoriatic patients treated with GT. For genotyping, droplet digital PCR was applied. The GSTM1 copy number was normalized to β-globin reference gene. In five GSTM1*1/*1 subjects, the GSTM1 to β-globin ratio moved from 0.99 to 1.03 with a median of 1.01. GSTM1*0/*1 heterozygotes (n = 20) contained only one GSTM1 function allele which conditioned the ratio 0.47-0.53 (median 0.50). GSTM1*0/*0 individuals (n = 21) showed no amplification of the null variants because of the large deletion in GSTM1. BPDE-DNA concentrations ranged from 1.8 to 66.3 ng/µg with a median of 12.3 ng/µg. GSTM1*0/*0 and GSTM1*0/*1 genotypes showed non-significantly higher concentrations of BPDE-DNA adducts than the GSTM1*1/*1 one (12.3 and 12.4 vs 7.8 ng/µg). The non-significant relationship between BPDE-DNA adducts and GSTM1 genotypes in psoriatic patients could be associated with relatively low doses of CCT and short-term UV-R exposures used in GT.

• MeSH
• adukty DNA MeSH
• dehet uhelný terapeutické užití   MeSH
• dospělí MeSH
• genotyp MeSH
• glutathiontransferasa genetika   MeSH
• keratolytika terapeutické užití   MeSH
• kombinovaná terapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutační analýza DNA metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus genetický MeSH
• psoriáza genetika   terapie   MeSH
• sekvence nukleotidů * MeSH
• sekvenční delece * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• terapie ultrafialovými paprsky MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Real-time quantitative PCR (qPCR) is a well-established tool for minimal residual disease (MRD) detection in mature lymphoid malignancies. Despite remarkable sensitivity and specificity, qPCR has some limitations, particularly in the need for a reference standard curve, based on target serial dilutions. In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. We then compared the two approaches in 69 patients with a documented molecular marker at diagnosis (18 multiple myelomas, 21 mantle cell lymphomas assessed with the immunoglobulin gene rearrangement, and 30 follicular lymphomas with the use of the BCL2/immunoglobulin gene major breakpoint region rearrangement). ddPCR was successful in 100% of cases, whereas qPCR failed to provide a reliable standard curve in three patients. Overall, 222 of 225 samples were evaluable by both methods. The comparison highlighted a good concordance (r = 0.94, P < 0.0001) with 189 of 222 samples (85.1%; 95% CI, 80.4%-89.8%) being fully concordant. We found that ddPCR is a reliable tool for MRD detection with greater applicability and reduced labor intensiveness than qPCR. It will be necessary to authorize ddPCR as an outcome predictor tool in controlled clinical settings and multilaboratory standardization programs.

• MeSH
• folikulární lymfom diagnóza   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• lymfom z plášťových buněk diagnóza   MeSH
• mnohočetný myelom diagnóza   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor diagnóza   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH