The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

• MeSH
• Genetic Therapy * MeSH
• Polymerase Chain Reaction MeSH
• Tissue Distribution MeSH
• Drug Development * MeSH
• Publication type
• Journal Article MeSH

The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.

• MeSH
• Biosensing Techniques * MeSH
• Polymerase Chain Reaction MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.

• Publication type
• Journal Article MeSH

Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.

• Publication type
• Journal Article MeSH

Digitalizace laboratoří, aplikace big dat a automatizovaná strojová diagnostika ("machine learning") jsou nástroji pro vznik a fungování toho, co se označuje jako precizní medicína. Genomika, její dominantní metody (qPCR, dPCR, ddPCR, NGS), produkující obrovská kvanta dat (big data) a schopnosti počítačových systémů tyto soubory dat využívat v diagnostice a terapii za významného přispění "umělé inteligence" se označují jako strojová automatizovaná diagnostika - machine learning respektive deep learning). Tyto postupy pronikají z průmyslu a výzkumu do rutinní medicíny včetně medicíny laboratorní. Zvládnutí technických a personálních problémů těchto změn bude stát značné úsilí, srovnatelné s před lety realizovanou přeměnou manuální laboratorní práce na automatizovanou činnost a s přeměnou papírové dokumentace výsledků na laboratorní a nemocniční informační systémy. Lze předpokládat nejen zásadní změny metod laboratorní práce, ale i změny požadavků na odbornost personálu laboratoří a rovněž lze předpokládat nevyhnutelnost radikálního ovlivnění činnosti klinických laboratoří. Etický rozměr nastávajících změn bude stejně závažný, jako ten technický a bude možné očekávat nejen významný progres v diagnostice e prognostice chorob, ale i vzestup rizika zdravotní péče v případě chyb a neprofesionality. Automatická strojová aplikace big dat a používání umělé inteligence jsou náročné, je s nimi v medicíně málo zkušeností, ale vyhnout se jim nebude možné.

Digitalization of clinical laboratories, application of big data and methods of machine learning re contemporary tools for precision medicine. Precision medicine is based mainly on the genomic methods, namely of dominant PCR and NGS methods. These methods produces enormous number of dates (big data) and can be explored by means of artificial intelligence in processes called machine learning. Machine learning was primarily used in industry and research and now contemporary penetrates into medicine and also to laboratory medicine. Methods based on the big data and artificial intelligence with exploration of big data is certainly very important factor of future of medicine. It will be needs large requirements not only on high-technology equipment, but also for new type of young laboratory Professional used basically new methods of work and mind. Machine learning, part of precision medicine, necessary namely for oncology and prediction of patients state crettemeans also lot of new types of ethical problems. These ethical questions and problems should be soluted immediately, parallel with introduction of machine learning to laboratory practice.

• MeSH
• Big Data MeSH
• Diagnosis, Computer-Assisted * methods   MeSH
• Precision Medicine MeSH
• Humans MeSH
• Machine Learning MeSH
• Artificial Intelligence MeSH
• Check Tag
• Humans MeSH

Vysokoškolská praktická cvičení, která se zaměřují na mikrobiologii.

• MeSH
• Microbiological Techniques MeSH
• Microbiology MeSH

• Conspectus
• Mikrobiologie
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• mikrobiologie, lékařská mikrobiologie
• NML Publication type
• učebnice vysokých škol
• praktická cvičení

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.

• Keywords
• digitální PCR,
• MeSH
• DNA MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Polymerase Chain Reaction classification   methods   MeSH
• Sensitivity and Specificity MeSH
• Nucleic Acid Amplification Techniques MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Současné výsledky léčby chronické myeloidní leukemie, dosahované pomocí tyrozinkinázových inhibitorů (TKI) jsou zcela excelentní, více jak 80 % nemocných diagnostikovaných v chronické fázi onemocnění přežívá deset let a více, vysoké je také procento dlouhodobých hlubokých molekulárních odpovědí. Aktuálními trendy usilujícími o další zlepšení výsledků léčby jsou možnost ukončit léčbu TKI při trvající molekulární odpovědi a volba individualizovaného přístupu k pacientovi. Zcela zásadní je také monitorování minimální zbytkové nemoci, jejíž přesnější kvantifikace pomocí digitální PCR na úrovni BCR-ABL1 transkriptu nebo na úrovni DNA, s cílem detekovat i transkripčně neaktivní leukemické buňky, může v blízké budoucnosti poskytovat lékaři při rozhodování o ukončení léčby spolehlivé vodítko.

Current CML therapy based on tyrosine kinase inhibitors (TKI) provides excellent outcomes: more than 80% of patients diagnosed in chronic phase of CML survive at least 10 years and a quite high number of patients have a long-term deep molecular response. Nevertheless, modern trends in clinical practice include discontinuation of TKI therapy in patients with sustained deep molecular response. Patient concerns and health care cost considerations have prompted research into the possibility of TKI treatment-free remission for selected patients or into the setting up individual treatment approaches based on the identification of disease progression markers. To apply this strategy, monitoring of minimal residual disease and early detection of relapse is absolutely critical. Digital PCR (dPCR) represents a very precise and sensitive technology for detecting BCR-ABL1 at mRNA or DNA level – with a capability of detecting CML cells lacking expression of BCR-ABL1. Exact measurement of minimal residual disease is key for CML therapy management and eventual TKI discontinuation.

• Keywords
• leukemická kmenová buňka,
• MeSH
• Drug Resistance, Neoplasm MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive * diagnosis   drug therapy   MeSH
• Genes, abl MeSH
• Protein Kinase Inhibitors * therapeutic use   MeSH
• Clinical Studies as Topic MeSH
• Pregnancy Complications MeSH
• Humans MeSH
• Mutation MeSH
• Biomarkers, Tumor MeSH
• Pregnancy Complications, Neoplastic MeSH
• Pregnancy MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Review MeSH