Bird sex determination is fundamental in various ecological and biological studies, although many avian species cannot be sexed visually due to their monomorphic and/or monochromatic appearance. Thus, reliable laboratory methods for sexing are a prerequisite. Most avian nestlings lack sex-related signs, including the Eurasian pygmy owl (Glaucidium passerinum). We performed laboratory sex determination analysis of this species using blood samples of 242 juveniles and nine adults. It relied on the qPCR of the specific intron from the chromo-helicase DNA-binding protein 1 gene. We tested three primer sets, the P2/P8, 2550F/2718R, and CHD1F/CHD1R, commonly used for bird laboratory sexing. The outcomes were displayed on an agarose gel electrophoresis and a plot from melt curve analysis, which had not been previously conducted in Eurasian pygmy owls. We found that only primer set CHD1F/CHD1R proved reliable, as the only one determined sex with one and two band/s and peak/s on the electrophoresis and the melt curve plot for males and females, respectively. The other two primer pairs failed and depicted one band/peak in all specimens regardless of their sex. Therefore, we recommend performing Eurasian pygmy owls' laboratory sexing by qPCR with CHD1F/CHD1R primers only.

• Klíčová slova
• 2550F/2718R, Birds of prey, CHD1F/CHD1R, Laboratory sexing, P2/P8, Sequencing,
• MeSH
• analýza určování pohlaví * metody   MeSH
• DNA primery * genetika   MeSH
• Stringiformes * genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA primery * MeSH

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.

• MeSH
• bronchoalveolární lavážní tekutina mikrobiologie   MeSH
• diagnostické techniky molekulární MeSH
• DNA fungální genetika   MeSH
• DNA primery genetika   MeSH
• houby genetika   izolace a purifikace   patogenita   MeSH
• imunokompromitovaný pacient * MeSH
• intergenová DNA genetika   MeSH
• invazivní mykotické infekce diagnóza   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• sekvenční analýza DNA MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA fungální MeSH
• DNA primery MeSH
• intergenová DNA MeSH

OBJECTIVES: Direct genotyping of adenovirus or enterovirus from clinical material using polymerase chain reaction (PCR) followed by Sanger sequencing is often difficult due to the presence of multiple virus types in a sample, or due to varying efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids. Here we present a simple protocol for virus genotyping using massive parallel amplicon sequencing. METHODS: The protocol utilized a set of 16 tailed degenerate primers flanking the seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent addition of dual indices enabled simultaneous sequencing of 384 different samples on an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on remapping to a set of references representative of the presently known repertoire of virus types. RESULTS: After validation with known virus types, the sequencing method was applied on 301 adenovirus-positive samples and 350 enterovirus-positive samples from a longitudinally collected series of stools from 83 children aged 3 to 36 months. We detected 7 different adenovirus types and 27 different enterovirus types. There were 37 (6.2%) samples containing more than one genotype of the same viral genus. At least one dual infection was experienced by 23 of 83 (28%) of the children observed over the 3 years' observation period. CONCLUSIONS: Amplicon sequencing with a multiplex set of degenerate primers seems to be a rapid and reliable technical solution for genotyping of large collections of samples where simultaneous infections with multiple strains can be expected.

• Klíčová slova
• adenovirus, enterovirus, genotype, infants, massive parallel sequencing, virus type,
• MeSH
• Adenoviridae klasifikace   genetika   izolace a purifikace   MeSH
• adenovirové infekce virologie   MeSH
• DNA primery genetika   MeSH
• enterovirové infekce virologie   MeSH
• Enterovirus klasifikace   genetika   izolace a purifikace   MeSH
• genotyp * MeSH
• genotypizační techniky metody   MeSH
• kojenec MeSH
• lidé MeSH
• longitudinální studie MeSH
• předškolní dítě MeSH
• sekvenční analýza DNA metody   MeSH
• výpočetní biologie MeSH
• zvířata MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Norsko MeSH
• Názvy látek
• DNA primery MeSH

• MeSH
• DNA primery genetika   MeSH
• genetické markery * MeSH
• hybridizace genetická * MeSH
• ryby klasifikace   genetika   MeSH
• sekvenční analýza DNA veterinární   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Rusko MeSH
• Názvy látek
• DNA primery MeSH
• genetické markery * MeSH

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P- and K-distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health-promoting probiotic bacteria.

• Klíčová slova
• Bifidobacteriaceae, Bifidobacterium, CTP synthetase, classification, phylogenetics, scardovia,
• MeSH
• Actinobacteria klasifikace   enzymologie   genetika   izolace a purifikace   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• DNA primery genetika   MeSH
• fylogeneze * MeSH
• ligasy tvořící vazby C-N chemie   genetika   metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• techniky typizace bakterií metody   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• CTP synthetase MeSH Prohlížeč
• DNA bakterií MeSH
• DNA primery MeSH
• ligasy tvořící vazby C-N MeSH
• RNA ribozomální 16S MeSH

Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen's kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.

• MeSH
• bakteriální geny genetika   MeSH
• DNA gyráza genetika   MeSH
• DNA primery genetika   MeSH
• endodeoxyribonukleasy genetika   MeSH
• mastitida skotu diagnóza   mikrobiologie   MeSH
• mléko mikrobiologie   MeSH
• Mycoplasma bovis genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• skot MeSH
• techniky amplifikace nukleových kyselin normy   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• DNA gyráza MeSH
• DNA primery MeSH
• endodeoxyribonukleasy MeSH
• RNA ribozomální 16S MeSH

The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (NT) or as 'K2:07', which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 NT and two biovar 1 'K2:O7' isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two 'K2:O7' isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the 'K2:O7' isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and 'K2:O7' isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously 'K2:O7'). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.

• Klíčová slova
• Actinobacillus pleuropneumoniae, Capsule genes, Diagnostics, PCR, Serovar 17, Serovar 18,
• MeSH
• Actinobacillus pleuropneumoniae klasifikace   genetika   imunologie   izolace a purifikace   MeSH
• bakteriální pouzdra genetika   MeSH
• DNA bakterií genetika   MeSH
• DNA primery genetika   MeSH
• genotyp * MeSH
• infekce bakteriemi rodu Actinobacillus epidemiologie   veterinární   MeSH
• nemoci prasat epidemiologie   mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• prasata MeSH
• sekvenování celého genomu MeSH
• séroskupina * MeSH
• sérotypizace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Dánsko epidemiologie   MeSH
• Kanada epidemiologie   MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA primery MeSH

After its computational inference from human stool metagenomes, the CrAssphage has proven to be the most prevalent phage in the human gut, with presumably very wide geographic distribution. The currently available molecular assays do not sufficiently reflect the CrAssphage sequence variability. Here, we report a novel real-time PCR assay whose primers and probes are derived from data of multiple CrAssphage strains obtained from gut viral metagenomes of European, Asian, and African subjects. This assay can be useful in analyses of putative bacterial host co-occurence, and in association studies of non-infectious diseases where the phage may modify the content of gut bacteriomes.

• Klíčová slova
• Africa, Asia, polymorphism, virome,
• MeSH
• bakteriofágy genetika   izolace a purifikace   MeSH
• DNA primery genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• mikrobiota * MeSH
• oligonukleotidové sondy genetika   MeSH
• střevní mikroflóra * MeSH
• virová nálož metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Afrika MeSH
• Asie MeSH
• Evropa MeSH
• Názvy látek
• DNA primery MeSH
• oligonukleotidové sondy MeSH

BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

• Klíčová slova
• High-throughput sequencing, PCR, Targeted resequencing, Unique molecular identifiers,
• MeSH
• DNA primery genetika   MeSH
• erbB receptory genetika   MeSH
• genová knihovna * MeSH
• polymerázová řetězová reakce metody   MeSH
• sekvenční analýza DNA MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA primery MeSH
• erbB receptory MeSH

Psittacine beak and feather disease (PBFD) is one of the most significant viral diseases in psittacine birds. The aim of the presented study was to develop a highly specific and sensitive TaqMan real-time PCR assay for universal detection of beak and feather disease virus (BFDV). Primers and a hydrolysis probe were selected on the highly conserved regions belonging to the ORF1 of the BFDV genome which were identified by aligning 814 genomic sequences downloaded from the GenBank database. The evaluation of the reaction parameters suggested a reaction efficiency of 97.1%, with consistent detection of 101 virus copies/μl of nucleic acid extract. The low values of standard deviation and coefficient of variation indicate a high degree of reproducibility and repeatability. The diagnostic applicability of the assay was proven on 36 BFDV positive and 107 negative specimens of psittacine origin representing 28 species. The assay showed a 100% ability to detect distinct genetic variants of the virus. Our data suggest that the presented TaqMan real-time PCR represents a specific, sensitive and reliable assay facilitating the molecular detection of BFDV.

• Klíčová slova
• Beak and feather disease virus (BFDV), Real-time PCR,
• MeSH
• Circovirus genetika   izolace a purifikace   MeSH
• diagnostické techniky molekulární metody   MeSH
• DNA primery genetika   MeSH
• infekce viry čeledi Circoviridae diagnóza   veterinární   virologie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• nemoci ptáků diagnóza   virologie   MeSH
• oligonukleotidové sondy genetika   MeSH
• ptáci MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• veterinární lékařství metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• DNA primery MeSH
• oligonukleotidové sondy MeSH