multiplexing
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Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.
Multiplex PCR is one of the many variants routinely performed by polymerase chain reaction (PCR). Its advantage is especially rapid detection of a large number of products in one reaction using multiple primer sets. Optimization of the multiplex PCR protocol is important also for genes of metallothionein isoforms, so that we can carry out the amplification of more than one DNA sequence. Necessary part of this proces is the adjustments in temperature during the reaction steps and also intervention with the changes in the concentration of individual reagents and different combinations or the amount of each primer.
This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.
- MeSH
- Actinomycetales genetika izolace a purifikace MeSH
- Ascomycota genetika izolace a purifikace MeSH
- Begomovirus genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Fusarium genetika izolace a purifikace MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci rostlin mikrobiologie virologie MeSH
- Solanum lycopersicum mikrobiologie virologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.
- MeSH
- bakteriální geny MeSH
- bakteriologické techniky metody normy MeSH
- DNA bakterií genetika MeSH
- DNA primery genetika MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody normy MeSH
- plošný screening metody normy MeSH
- Streptococcus agalactiae klasifikace genetika izolace a purifikace MeSH
- Check Tag
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells.
- MeSH
- azidy chemie MeSH
- biofilmy * MeSH
- Campylobacter genetika izolace a purifikace fyziologie MeSH
- interkalátory chemie MeSH
- mikrobiální viabilita * MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- propidium analogy a deriváty chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
- MeSH
- infekce yersiniemi diagnóza mikrobiologie MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci přenášené potravou diagnóza mikrobiologie parazitologie MeSH
- Toxoplasma genetika izolace a purifikace MeSH
- toxoplazmóza diagnóza parazitologie MeSH
- Yersinia enterocolitica genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Je popsána kazuistika 30letého muže, který byl odeslán do akné poradny pro kožní obtíže trvající asi 8 let. Objektivně byla na obličeji, krku a na trupu přítomna četná vyklenutí, největší pod levým obočím a pod pravým boltcem. Na trupu byly ojedinělé komedony, papuly a noduly. Histologické vyšetření jedné z cyst pod pravým boltcem stanovilo diagnózu steatocystoma multiplex. Pro současné diagnózy acne nodulocystica a steatocystoma multiplex byla zahájena léčba perorálním izotretinoinem v počáteční dávce 0,25 mg/kg/den, po měsíci byla dávka navýšena na 0,40 mg/kg/den. Akné byla zhojena po 6 měsících, pro steatocystoma multiplex bylo v léčbě pokračováno. Od 20. měsíce byla dávka izotretinoinu postupně snížována až na 0,075 mg/kg/den (10 mg 4× týdně). Pacient toleruje tuto léčbu velmi dobře. Lokálně po celou dobu aplikuje adapalen v krému na noc. Po 1,5 roce byla excidována největší cysta pod levým obočím s velmi dobrým kosmetickým efektem. Pacient je v péči akné poradny již 3 roky. Cystičky na obličeji, krku a na trupu se pomalu zmenšují, některé vymizely. Vzhledem k chronicitě diagnózy steatocystoma multiplex je u pacienta plánována léčba malými dávkami perorálního izotretinoinu dlouhodobě za pravidelných kontrol. Pacient je s efektem léčby velmi spokojený.
A casuistics of a 30-year-old man who was sent to our Acne Clinic because of skin problems lasting for about 8 years is presented. Objectivelly, on the face, neck and trunk many arches were present, the biggest one under the left eyebrow and under the right ear. There were some comedones, papules and small nodules on the trunk. Histological examination of a cyst under the right ear revealed a diagnosis of steatocystoma. Because of concomitant diagnoses acne nodulocystica and steatocystoma multiplex, a treatment with peroral isotretinoin was started with an initial dose 0,25 mg/kg/day, after one month the dose was increased to 0,40 mg/kg/day. Acne was cleared within 6 months. Because of steatocystoma multiplex, the treatment with peroral isotretinoin has been continued. After 20 months of the treatment, the dose of isotretinoin was decreased step by step to a dose 0,075 mg/kg/den (10 mg 4× weekly). The treatment is very well tolerated. For the whole time, local adapalen cream in the evening is applied. In the meantime, the biggest cyst under the left eyebrow was excised with a very good cosmetic result. Cysts on the face, neck and trunk have been slowly diminishing, some have disappeared . Because of steatocystoma multiplex is a chronic disease, a long- term treatment with a low- dose of peroral isotretinoin under regular check-ups has been planned. The patient is very satisfied with the treatment ́s effect.
- MeSH
- acne vulgaris farmakoterapie MeSH
- aplikace orální MeSH
- dermatologické látky MeSH
- dospělí MeSH
- genetické nemoci vrozené diagnóza terapie MeSH
- isotretinoin aplikace a dávkování terapeutické užití MeSH
- lidé MeSH
- steatocystoma multiplex * chirurgie diagnóza farmakoterapie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.
- MeSH
- fluorescenční barviva * MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- multiplexová polymerázová řetězová reakce přístrojové vybavení metody MeSH
- ptačí chřipka u ptáků diagnóza virologie MeSH
- ptáci MeSH
- virus chřipky A klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.
- MeSH
- Actinobacillus pleuropneumoniae klasifikace genetika patogenita MeSH
- bakteriální polysacharidy genetika MeSH
- bakteriální pouzdra chemie klasifikace genetika MeSH
- infekce bakteriemi rodu Actinobacillus diagnóza mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- nemoci prasat diagnóza mikrobiologie MeSH
- prasata MeSH
- sekvenční analýza * MeSH
- séroskupina MeSH
- sérotypizace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH