Sekvenování genů 16S, 18S a ITS regionů se stalo jedním z nejdůležitějších nástrojů v molekulární diagnostice, zejména v oblasti mikrobiologie, patologie a soudního lékařství. Výše zmíněné geny, obsahující konzervované i variabilní oblasti, jsou hojně využívány pro taxonomické zařazení bakterií a eukaryot. Sekvenování 16S rDNA umožňuje detekci bakteriálních infekcí, zatímco sekvenace ITS regionů a 18S rDNA je využívána při identifikaci mykotických, případně parazitárních infekcí, a to především v případech, kdy tradiční metody selhávají. Tento článek se zaměřuje na rozšířené možnosti těchto metod, jejich uplatnění v klinické diagnostice a výzkumu, zkoumá výhody a nevýhody, a diskutuje potenciální budoucí vývoj v oblasti technologie sekvenování nové generace (NGS).
Gene sequencing of 16S, 18S, and ITS regions is a crucial tool in molecular diagnostics, especially in microbiology, pathology and forensic medicine. These genes contain conserved and variable regions and are widely used for the taxonomic classification of bacteria and eukaryotes. Sequencing of 16S rDNA helps detect bacterial infections, while sequencing of ITS regions and 18S rDNA is used to identify fungal or parasitic infections, especially when traditional methods are ineffective. This article focuses on the expanded possibilities of these methods, their application in clinical diagnostics and research, their advantages and disadvantages, and discusses potential future developments in the field of next-generation sequencing (NGS) technology.
- Klíčová slova
- Internal Transcribed Spacers, 16S rDNA, 18S rDNA,
- MeSH
- diagnostické techniky molekulární klasifikace metody MeSH
- DNA bakterií analýza genetika klasifikace MeSH
- DNA analýza genetika klasifikace ultrastruktura MeSH
- infekční nemoci * diagnóza genetika MeSH
- lidé MeSH
- vysoce účinné nukleotidové sekvenování * klasifikace metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.
- MeSH
- esenciální geny * MeSH
- malá jadérková RNA metabolismus MeSH
- posttranskripční úpravy RNA * MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA ribozomální 18S metabolismus MeSH
- Trypanosoma brucei brucei enzymologie metabolismus MeSH
- umlčování genů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This paper reports results of the first cytogenetic study carried out on a recently described monozoic tapeworm, Khawia saurogobii Xi et al., 2009, from the Chinese lizard gudgeon (Saurogobio dabryi). The karyotype of this species is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16; n = 3m + 5t), metacentric chromosomes representing the first, sixth, and eight pairs. All chromosomes except the largest pair displayed 4',6-diamidino-2-phenylidole (DAPI) positive heterochromatin in centromeric regions. In mitotic preparations stained with Giemsa, one of the homologues of a smaller metacentric chromosome pair (No. 7) showed a distinct secondary constriction, whereas the other did not. Fluorescent in situ hybridization (FISH) with 18S ribosomal DNA (rDNA) probe revealed that the chromosomes No. 7 carry each a cluster of ribosomal genes associated with the centromeric heterochromatin and confirmed that this chromosome pair contains a nucleolar organizer region (NOR). The rDNA-FISH also confirmed heteromorphism in the size of NOR (i.e., secondary constriction) observed after Giemsa staining. The present cytogenetic analysis revealed species-specific characters of K. saurogobii and showed that FISH may represent a new valuable cytogenetic tool suitable for comparative taxonomic or phylogenetic studies within the order Caryophyllidea in the future.
- MeSH
- barvení a značení MeSH
- Cestoda genetika MeSH
- chromozomy MeSH
- cytogenetické vyšetření MeSH
- heterochromatin metabolismus MeSH
- hybridizace in situ fluorescenční MeSH
- indoly metabolismus MeSH
- karyotypizace MeSH
- mapování chromozomů MeSH
- organizátor jadérka MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 18S genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89-95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73-5.04 μm) × 3.94 μm (3.50-4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships.
- MeSH
- aktiny genetika MeSH
- Cryptosporidium genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genotyp MeSH
- kryptosporidióza epidemiologie MeSH
- molekulární sekvence - údaje MeSH
- prevalence MeSH
- proteiny tepelného šoku HSP70 genetika MeSH
- RNA ribozomální 18S genetika MeSH
- Sciuridae parazitologie MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Ectoparasitic monogeneans of the family Diplozoidae have direct and monoxenous life cycle. The cytogenetics of monogeneans in general and diplozoids in particular, is a relatively underexplored area. This is why each new detailed description of a karyotype provides significant information about the evolution of monogenean chromosomes and contributes to a better understanding of phylogenetic relationships within this group. This study offers new data on the chromosomes of Eudiplozoon nipponicum, an invasive parasite of the common carp. This species' karyotype consists of seven pairs of telocentric chromosomes (2n = 14 t). After DAPI staining, we marked heterochromatin blocks on all chromosomes in the pericentromeric region. Silver staining (AgNO3) and staining with fluorescent dye YOYO-1 revealed the presence of one large active nucleolus. Fluorescent in situ hybridization with an 18S rDNA probe revealed one cluster of ribosomal genes at the terminal part of the long arms of chromosome pair No. 7. We compared our results with studies on the phylogenetic relationships of diplozoids which applied a combination of molecular methods and classical morphological characterization and found that the results of our cytogenetic analysis are consistent with the hypothesis that E. nipponicum is more basal member of the family Diplozoidae.
- MeSH
- barvení a značení MeSH
- buněčné dělení * MeSH
- cytogenetické vyšetření MeSH
- fylogeneze MeSH
- hybridizace in situ fluorescenční MeSH
- kapři parazitologie MeSH
- karyotyp MeSH
- RNA ribozomální 18S genetika MeSH
- spermatocyty fyziologie MeSH
- Trematoda genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory.
- MeSH
- Babesia genetika MeSH
- babezióza krev diagnóza parazitologie MeSH
- fylogeneze MeSH
- genetická variace * MeSH
- genetické markery MeSH
- genom protozoální MeSH
- genotyp * MeSH
- haplotypy MeSH
- kohortové studie MeSH
- mitochondrie genetika MeSH
- nemoci psů diagnóza parazitologie MeSH
- protozoální DNA genetika MeSH
- psi MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa MeSH
- MeSH
- DNA helmintů analýza MeSH
- Dracunculus (Nematoda) genetika klasifikace MeSH
- finanční podpora výzkumu jako téma MeSH
- fylogeneze MeSH
- hlístice genetika klasifikace MeSH
- lidé MeSH
- ribozomální DNA analýza MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Free ranging ungulates, represented in Europe mostly by several deer species, are important hosts for ticks and reservoirs of tick-borne infections. A number of studies have focused on the prevalence of tick borne pathogens in deer chiefly with the aim to determine their potential role as reservoir hosts for important human and livestock pathogens. However, genetic similarity of Babesia spp. forming a group commonly termed as a clade VI that accommodates the deer piroplasms, complicates this task and has led to the description of a bewildering array of poorly characterised strains. This study aims to resolve this issue by using two independent genetic loci, nuclear 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes, used in parallel to identify Babesia isolates in free-ranging red, sika, and roe deer in two areas of their co-occurrence in the Czech Republic. The COX1 loci, in contrast to 18S rRNA gene, shows a clear difference between interspecific and intraspecific variation at the nucleotide level. The findings confirm B. divergens, Babesia sp. EU1 and B. capreoli in studied deer species as well as common presence of another unnamed species that matches a taxon previously referred to as Babesia sp. or Babesia cf. odocoilei or Babesia CH1 group in several other sites throughout Europe. The invasive sika deers enter the life cycle of at least three piroplasmid species detected in native deer fauna. The presence of B. divergens in both sika and red deer in an area where bovine babesiosis is apparently absent raises important questions regarding the epidemiology, host specificity and taxonomic status of the parasite.
- MeSH
- Babesia klasifikace genetika MeSH
- babezióza parazitologie virologie MeSH
- cyklooxygenasa 1 genetika MeSH
- fylogeneze MeSH
- molekulární evoluce MeSH
- protozoální DNA genetika MeSH
- protozoální proteiny genetika MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční analýza DNA metody MeSH
- vysoká zvěř parazitologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Metacestodes of Mesocestoides sp. were recorded from Sturnus vulgaris (Passeriformes: Stumidae) in the Czech Republic in April 2002. They were found in a cutaneous cyst and in the thoracic region of the body cavity of the bird. This is the first record of metacestodes of Mesocestoides sp. in this host species in Europe as well as the first finding of the formation of a cutaneous cyst provoked by this parasite. Additional specimens from Apodemus agrarius (Mammalia: Rodentia) from Bulgaria and Lacerta agilis (Reptilia: Squamata) from the Czech Republic were compared with that from S. vulgaris. Sequence data from the V4 variable region (18S rDNA) were used to compare genetic variability among these and previously characterized isolates of Mesocestoides spp. A number of distinct clades were recognized, with metacestodes from L. agilis showing the highest degree of relative divergence.
- MeSH
- Cestoda anatomie a histologie genetika izolace a purifikace MeSH
- DNA helmintů chemie genetika MeSH
- fylogeneze MeSH
- hrudník parazitologie MeSH
- kůže parazitologie MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- RNA ribozomální 18S chemie genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- zpěvní ptáci parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
