The house mouse, Mus musculus, is a widely used animal model in biomedical research, with classical laboratory strains (CLS) being the most frequently employed. However, the limited genetic variability in CLS hinders their applicability in evolutionary studies. Wild-derived strains (WDS), on the other hand, provide a suitable resource for such investigations. This study quantifies genetic and phenotypic data of 101 WDS representing 5 species, 3 subspecies, and 8 natural Y consomic strains and compares them with CLS. Genetic variability was estimated using whole mtDNA sequences, the Prdm9 gene, and copy number variation at two sex chromosome-linked genes. WDS exhibit a large natural variation with up to 2173 polymorphic sites in mitogenomes, whereas CLS display 92 sites. Moreover, while CLS have two Prdm9 alleles, WDS harbour 46 different alleles. Although CLS resemble M. m. domesticus and M. m. musculus WDS, they differ from them in 10 and 14 out of 16 phenotypic traits, respectively. The results suggest that WDS can be a useful tool in evolutionary and biomedical studies with great potential for medical applications.

• MeSH
• alely MeSH
• divoká zvířata genetika   MeSH
• druhová specificita MeSH
• fenotyp MeSH
• genetická variace * MeSH
• histonlysin-N-methyltransferasa genetika   MeSH
• mitochondriální DNA genetika   MeSH
• myši * genetika   MeSH
• variabilita počtu kopií segmentů DNA MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši * genetika   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histonlysin-N-methyltransferasa MeSH
• mitochondriální DNA MeSH
• prdm9 protein, mouse MeSH Prohlížeč

This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 μm to 5 μm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.

• Klíčová slova
• Electrical and electronic engineering, Microfluidics,
• Publikační typ
• časopisecké články MeSH

Microfluidics systems can be fabricated in various ways using original silicon glass systems, with easy Si processing and surface modifications for subsequent applications such as cell seeding and their study. Fluorescent imaging of cells became a standard technique for the investigation of cell behavior. Unfortunately, high sensitivity fluorescent imaging, e.g., using total internal reflection fluorescence (TIRF) microscopy, is problematic in these microfluidic systems because the uneven surfaces of the silicon channels' bottoms affect light penetration through the optical filters. In this work, we study the nature of the phenomenon, finding that the problem can be rectified by using a silicon-on-insulator (SOI) substrate, defining the channel depth by the thickness of the top Si layer, and halting the etching at the buried SiO2 layer. Then the fluorescent background signal drops by = 5 times, corresponding to the limit of detection drop from = 0.05 mM to = 50 nM of fluorescein. We demonstrate the importance of a flat surface using TIRF-based single-molecule detection, improving the signal to a noise ratio more than 18 times compared to a conventional Si wafer. Overall, using very high-quality SOI substrates pays off, as it improves the fluorescence image quality due to the increase in signal-to-noise ratio. Concerning the cost of microfluidic device fabrication-design, mask fabrication, wafer processing, and device testing-the initial SOI wafer cost is marginal, and using it improves the system performance.

• MeSH
• křemík * chemie   MeSH
• mikrofluidika * MeSH
• nanotechnologie metody   MeSH
• oxid křemičitý MeSH
• poměr signál - šum MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• křemík * MeSH
• oxid křemičitý MeSH

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (T M). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured T M values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured T M as the parameter. Even when the actual T M of the PCR solution was constant, the measured T M value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known T M as a temperature sensor. Among the non-contact temperature measurement methods, the proposed T M-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on T M allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.

• Publikační typ
• časopisecké články MeSH

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

• Publikační typ
• časopisecké články MeSH

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

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

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

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

PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

• Klíčová slova
• CDCE, constant denaturing capillary electrophoresis, Chimera, DGGE, denaturing gradient gel electrophoresis, DHPLC, denaturing high-performance liquid chromatography, HPLC, high-performance liquid chromatography, Multi-template PCR, PAAG, polyacrylamide gel, SSCA, single strand conformation analysis, T-RFLP, terminal restriction fragment length polymorphism, TGGE, temperature gradient gel electrophoresis,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH