• Klíčová slova
• Candida, Candida parapsilosis complex, MALDI-TOF, fluorescent capillary electrophoresis, fungi,
• MeSH
• Candida parapsilosis klasifikace   izolace a purifikace   MeSH
• DNA fungální genetika   MeSH
• elektroforéza kapilární metody   normy   MeSH
• fluorescence MeSH
• intergenová DNA genetika   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• intergenová DNA MeSH

Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.

• Klíčová slova
• 16S rDNA, DNA extraction method, ITS rDNA, fungal microbiota, gut microbiome, gut microbiota, gut mycobiome, gut mycobiota,
• Publikační typ
• časopisecké články MeSH

Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize Candida sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ITS2 ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different Candida species were detected, with Candida parapsilosis (31.4%), Candida albicans (26.5%), and Candida tropicalis (12.4%) predominating. f-ITS2-PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 µl of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f-ITS2-PCR-CE after the inoculation volume was increased to 100 µl in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f-ITS2-PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.

• Klíčová slova
• Candida, biofilms, capillary electrophoresis, fungi, panfungal PCR, polyfungal sample, ureteral stents, urinary catheters,
• MeSH
• biofilmy růst a vývoj   MeSH
• Candida albicans izolace a purifikace   MeSH
• Candida parapsilosis izolace a purifikace   MeSH
• Candida tropicalis izolace a purifikace   MeSH
• Candida klasifikace   izolace a purifikace   MeSH
• diagnostické techniky molekulární přístrojové vybavení   metody   MeSH
• DNA fungální genetika   MeSH
• elektroforéza kapilární metody   MeSH
• fluorescence MeSH
• kandidóza mikrobiologie   moč   MeSH
• lidé MeSH
• močové katétry mikrobiologie   MeSH
• počet mikrobiálních kolonií normy   MeSH
• ribozomální DNA genetika   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• stenty mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• ribozomální DNA MeSH

High resolution melting analysis (HRMA) is a single-tube method, which can be carried out rapidly as an additional step following real-time quantitative PCR (qPCR). The method enables the differentiation of genetic variation (down to single nucleotide polymorphisms) in amplified DNA fragments without sequencing. HRMA has previously been adopted to determine variability in the amplified genes of a number of organisms. However, only one work to date has focused on pathogenic parasites-nematodes from the genus Trichinella. In this study, we employed a qPCR-HRMA assay specifically targeting two sequential gene fragments-cytochrome c oxidase subunit I (COI) and expansion segment V (ESV), in order to differentiate 37 single L1 muscle larvae samples of eight Trichinella species. We show that qPCR-HRMA based on the mitochondrial COI gene allows differentiation between the sequences of PCR products of the same length. This simple, rapid and reliable method can be used to identify at the species level single larvae of eight Trichinella taxa.

• MeSH
• denaturace nukleových kyselin MeSH
• genotypizační techniky metody   normy   MeSH
• polymorfismus genetický MeSH
• respirační komplex IV genetika   MeSH
• taxonomické DNA čárové kódování metody   normy   MeSH
• Trichinella klasifikace   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• respirační komplex IV MeSH

Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex.

• MeSH
• Candida izolace a purifikace   MeSH
• časové faktory MeSH
• DNA fungální chemie   genetika   MeSH
• elektroforéza kapilární metody   MeSH
• kandidóza diagnóza   MeSH
• lidé MeSH
• mezerníky ribozomální DNA chemie   genetika   MeSH
• mikrobiologické techniky metody   MeSH
• polymerázová řetězová reakce metody   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• DNA fungální MeSH
• mezerníky ribozomální DNA MeSH