polymerase-chain reaction
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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.
In a pilot study Double Repetitive Element-Polymerase Chain Reaction 20 clinical isolates of Mycobacterium tuberculosis from Guatemala and 49 strains from Prague were typed. This technique is based on direct evidence of repetitive elements IS6110 or PGRS and does not require DNA purification, digestion by endonuclease nor Southern blot hybridization. Preliminary examination of Guatemalian strains revealed a striking identity or similarity of DRE-PCR profiles while the Prague strains were characterized by conspicuous polymorphism. The Prague strains were examined in a total number of 13 series of electrophoreograms and subsequently subjected to automated analysis with GelCompar software. The DRE-PCR method is suitable for screening of a major number of clinical isolates of M. tuberculosis in laboratories equipped with a minimum of technical facilities for the PCR method, furthermore it requires facilities for synthesis of the necessary primers and at least basic knowledge of molecular biology.
- MeSH
- DNA bakterií genetika MeSH
- DNA fingerprinting MeSH
- lidé MeSH
- molekulární epidemiologie MeSH
- Mycobacterium tuberculosis klasifikace genetika MeSH
- plicní tuberkulóza epidemiologie mikrobiologie MeSH
- polymerázová řetězová reakce * MeSH
- repetitivní sekvence nukleových kyselin * MeSH
- techniky typizace bakterií * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
- Guatemala epidemiologie MeSH
- Názvy látek
- DNA bakterií MeSH
The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.
- Klíčová slova
- Absolute quantification, Digital polymerase chain reaction, Duplexing methods, Single fluorescence channel duplexing,
- MeSH
- barvicí látky * MeSH
- polymerázová řetězová reakce MeSH
- vyšetření u lůžka * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- barvicí látky * MeSH
Simple and reliable procedure for sample preparation and reverse transcription-polymerase chain reaction (RT-PCR) detection of potato virus A (PVA) is described. PVA-specific primers used in the RT-PCR defined a target sequence of 321 bp and did not produce amplification product(s) with potato virus Y.
Conditions for assessing KM-19 probe detected by Pst-1 restriction fragment length polymorphism (RFLP) by means of polymerase chain reaction were provided. Computer controlled mechanical arm with waterbaths and cloned heat-stable DNA polymerase was used. Results of KM-19 allelic frequencies on 90 cystic fibrosis chromosomes are presented. Allele two frequency was -0.833.
- MeSH
- alely MeSH
- cystická fibróza diagnóza genetika MeSH
- DNA genetika MeSH
- frekvence genu MeSH
- genetické markery MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- prenatální diagnóza MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- genetické markery MeSH
A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.
- MeSH
- DNA bakterií analýza MeSH
- feces mikrobiologie MeSH
- kultivační média MeSH
- kur domácí mikrobiologie MeSH
- maso mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- Salmonella genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- kultivační média MeSH
The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.
- MeSH
- Bifidobacterium genetika MeSH
- DNA bakterií izolace a purifikace MeSH
- Lactobacillus genetika MeSH
- polymerázová řetězová reakce metody MeSH
- železité sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- ferrite MeSH Prohlížeč
- železité sloučeniny MeSH
Model samples of Campylobacter jejuni for polymerase chain reaction (PCR) were prepared by rapid and simple procedures consisting of centrifugation, proteinase K treatment, Chelex 100 treatment, and boiling lyses. A PCR based on specific amplification of the variable sequence of 16S rRNA gene was performed using Tth DNA polymerase and the PCR products were visualized by agarose gel electrophoresis. The assay allowed the detection of 10 CFU/mL C. jejuni in the physiological saline and 100 CFU/mL in the basic Park and Sanders broth.
- MeSH
- Campylobacter coli klasifikace genetika izolace a purifikace MeSH
- Campylobacter jejuni klasifikace genetika izolace a purifikace MeSH
- Campylobacter klasifikace genetika izolace a purifikace MeSH
- chlorid sodný MeSH
- kultivační média MeSH
- lidé MeSH
- polymerázová řetězová reakce metody MeSH
- potravinářská mikrobiologie * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorid sodný MeSH
- kultivační média MeSH
Polymerase chain reaction (PCR) techniques have been developed for the detection of microsporidian DNA in different biological samples. We used sequence data of the rRNA gene for the identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. cuniculi, and E. hellem in different biological samples of HIV-infected patients by PCR, Southern blot hybridization, restriction endonuclease digestion analysis, cloning, and comparative genetic sequencing. One primer pair was used for amplification of the entire small subunit (SSU)-rRNA gene of E. bieneusi, E. intestinalis, and E. hellem from samples with electron microscopy confirmed infection. The amplified 1.2 kb SSU-rRNA gene fragments were ligated into a pMOSBlue T-vector, transfected into pMOSBlue competent cells, and were used as positive controls. Several primer pairs and hybridization probes were used to amplify and identify microsporidian DNA from different samples. Light microscopical examination of samples was performed in all patients and transmission electron microscopy was done on a subset of patient samples. DNA products were obtained from all samples with confirmed microsporidial infections. The identity of the DNA fragments was determined by Southern blot hybridization or by restriction endonuclease digestion analysis or by DNA sequencing. The results show that PCR is a reliable and sensitive indicator for the presence of microsporidian DNA in different biological samples of HIV-infected patients. PCR can be used further for species differentiation of microsporidia, even between species which cannot be differentiated by light and/or electron microscopy.
- MeSH
- HIV infekce komplikace MeSH
- lidé MeSH
- Microsporida klasifikace genetika MeSH
- mikrosporidióza diagnóza MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce * MeSH
- protozoální DNA analýza MeSH
- RNA ribozomální genetika MeSH
- sekvence nukleotidů MeSH
- Southernův blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- protozoální DNA MeSH
- RNA ribozomální MeSH
