PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.

• Klíčová slova
• COVID-19, PCR, RNA virus diagnoses, digital PCR, microfluidics, point-of-care diagnostics, portable systems, reverse transcription PCR,
• MeSH
• Betacoronavirus genetika   izolace a purifikace   MeSH
• COVID-19 MeSH
• design vybavení MeSH
• klinické laboratorní techniky přístrojové vybavení   metody   MeSH
• koronavirové infekce diagnóza   virologie   MeSH
• lidé MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• pandemie MeSH
• polymerázová řetězová reakce přístrojové vybavení   metody   MeSH
• SARS-CoV-2 MeSH
• testování na COVID-19 MeSH
• virová pneumonie diagnóza   virologie   MeSH
• vyšetření u lůžka MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

• Klíčová slova
• COVID-19 diagnoses, Future of PCR, Microfluidics, Miniaturization, Point of care, Polymerase chain reaction,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Optofluidic devices combining optics and microfluidics have recently attracted attention for biomolecular analysis due to their high detection sensitivity. Here, we show a silicon chip with tubular microchannels buried inside the substrate featuring temperature gradient (∇T) along the microchannel. We set up an optical fluorescence system consisting of a power-modulated laser light source of 470 nm coupled to the microchannel serving as a light guide via optical fiber. Fluorescence was detected on the other side of the microchannel using a photomultiplier tube connected to an optical fiber via a fluorescein isothiocyanate filter. The PMT output was connected to a lock-in amplifier for signal processing. We performed a melting curve analysis of a short dsDNA - SYBR Green I complex with a known melting temperature (TM) in a flow-through configuration without gradient to verify the functionality of the proposed detection system. We then used the segmented flow configuration and measured the fluorescence amplitude of a droplet exposed to ∇T of ≈ 2.31 °C mm-1, determining the heat transfer time as ≈ 554 ms. The proposed platform can be used as a fast and cost-effective system for performing either MCA of dsDNAs or for measuring protein unfolding for drug-screening applications.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The global risk of viral disease outbreaks emphasizes the need for rapid, accurate, and sensitive detection techniques to speed up diagnostics allowing early intervention. An emerging field of microfluidics also known as the lab-on-a-chip (LOC) or micro total analysis system includes a wide range of diagnostic devices. This review briefly covers both conventional and microfluidics-based techniques for rapid viral detection. We first describe conventional detection methods such as cell culturing, immunofluorescence or enzyme-linked immunosorbent assay (ELISA), or reverse transcription polymerase chain reaction (RT-PCR). These methods often have limited speed, sensitivity, or specificity and are performed with typically bulky equipment. Here, we discuss some of the LOC technologies that can overcome these demerits, highlighting the latest advances in LOC devices for viral disease diagnosis. We also discuss the fabrication of LOC systems to produce devices for performing either individual steps or virus detection in samples with the sample to answer method. The complete system consists of sample preparation, and ELISA and RT-PCR for viral-antibody and nucleic acid detection, respectively. Finally, we formulate our opinions on these areas for the future development of LOC systems for viral diagnostics.

• Klíčová slova
• Commercialization, Immunoassays, LOC, Microfluidic, Nucleic acid amplification, Viral detection,
• MeSH
• biosenzitivní techniky MeSH
• design vybavení MeSH
• DNA virů analýza   MeSH
• ELISA MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• laboratoř na čipu * MeSH
• lidé MeSH
• mikrofluidní analytické techniky přístrojové vybavení   MeSH
• nukleové kyseliny analýza   MeSH
• virové nemoci diagnóza   MeSH
• vyšetření u lůžka MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA virů MeSH
• nukleové kyseliny MeSH

During infectious disease outbreaks, the centers for disease control need to monitor particular areas. Considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (POC) diagnostics, which could also create an Internet of Things (IoT) for healthcare via a global network. However, at present IoT based on a functional POC instrument is not available. Here we show a fast, user-friendly, and affordable IoT system based on a miniaturized polymerase chain reaction device. We demonstrated the system's capability by amplification of complementary deoxyribonucleic acid (cDNA) of the dengue fever virus. The resulting data were then automatically uploaded via a Bluetooth interface to an Android-based smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. The IoT system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by DNA or ribonucleic acid.

• Klíčová slova
• Dengue fever, Infectious diseases, IoT, PCR,
• Publikační typ
• časopisecké články MeSH

Nucleic acid amplification for the detection of infectious diseases, food pathogens, or assessment of genetic disorders require a laboratory setting with specialized equipment and technical expertise. Isothermal deoxyribonucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), exhibit characteristics ideal for point-of-care (POC) applications, since their instrumentation is simpler in comparison with the standard method of polymerase chain reaction. Other key advantages of LAMP are robustness and the production of pyrophosphate in the presence of the target gene, enabling to detect the reaction products using the naked eye. Polymerase inhibitors, presented in clinical samples, do not affect the amplification process, making LAMP suitable for a simple sample-to-answer diagnostic systems with simplified sample preparation. In this review, we discuss the trends in miniaturized LAMP techniques, such as microfluidic, paper-based, and digital with their advantages and disadvantages, especially for POC applications alongside our opinion of the future development of miniaturized LAMP.

• Klíčová slova
• Digital LAMP, LAMP, Microfluidic, Multi gene detection, PCR, POC, Paper-based LAMP, Single gene detection,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH