Wilt (Fusarium oxysporum f. sp. lentis; Fol) is one of the major diseases of lentil worldwide. Two hundred and thirty-five isolates of the pathogen collected from 8 states of India showed substantial variations in morphological characters such as colony texture and pattern, pigmentation and growth rate. The isolates were grouped as slow (47 isolates), medium (118 isolates) and fast (70 isolates) growing. The macroconidia and microconidia (3.0-77.5 × 1.3-8.8 μm for macroconidia and 1.8-22.5 × 0.8-8.0 μm for microconidia for length × width) were variable in size and considering the morphological features, the populations were grouped into 12 categories. Seventy representative isolates based on their morphological variability and place of origin were selected for further study. A set of 10 differential genotypes was identified for virulence analysis and based on virulence patterns on these 10 genotypes, 70 Fol isolates were grouped into 7 races. Random amplified polymorphic DNA (RAPD), universal rice primers (URPs), inter simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) were used for genetic diversity analysis. URPs, ISSR and SRAP markers gave 100% polymorphism while RAPD gave 98.9% polymorphism. The isolates were grouped into seven clusters at genetic similarities ranging from 21 to 80% using unweighted paired group method with arithmetic average analysis. The major clusters include the populations from northern and central regions of India in distinct groups. All these three markers proved suitable for diversity analysis, but their combined use was better to resolve the area specific grouping of the isolates. The sequences of rDNA ITS and TEF-1α genes of the representative isolates were analysed. Phylogenetic analysis of ITS region grouped the isolates into two major clades representing various races. In TEF-1α analysis, the isolates were grouped into two major clades with 28 isolates into one clade and 4 remaining isolates in another clade. The molecular groups partially correspond to the lentil growing regions of the isolates and races of the pathogen.
During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.
- MeSH
- bakteriální geny * MeSH
- denaturace nukleových kyselin MeSH
- DNA bakterií chemie genetika MeSH
- DNA primery MeSH
- epidemický výskyt choroby MeSH
- Escherichia coli klasifikace genetika izolace a purifikace MeSH
- genom bakteriální MeSH
- genotypizační techniky * MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- jednonukleotidový polymorfismus * MeSH
- multilokusová sekvenční typizace metody MeSH
- počítačová simulace MeSH
- polymerázová řetězová reakce metody MeSH
- repetitivní sekvence nukleových kyselin MeSH
- sekvenování celého genomu MeSH
- surveillance populace MeSH
- techniky typizace bakterií * MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
- MeSH
- Bacteria enzymologie genetika MeSH
- Bacteroidetes enzymologie genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Cas proteiny metabolismus MeSH
- CRISPR-Cas systémy * MeSH
- dimerizace MeSH
- DNA primery biosyntéza MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- Escherichia coli metabolismus MeSH
- exprese genu MeSH
- fylogeneze MeSH
- mutace MeSH
- prokaryotické buňky metabolismus MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.
- MeSH
- Bacteria enzymologie genetika izolace a purifikace MeSH
- bakteriologické techniky * MeSH
- beta-laktamasy genetika metabolismus MeSH
- beta-laktamová rezistence genetika MeSH
- DNA bakterií genetika MeSH
- DNA primery MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
At present, the traditional methods for the screening of Clostridium butyricum are not sufficiently selective and efficient. Therefore, it is necessary to establish a targeted and efficient screening method for the detection of C. butyricum. Bioinformatics was used in this study to find C. butyricum specific genes, and species-specific primers were designed based on the conserved regions of the targeted genes, followed by optimization of the PCR conditions. Methodological evaluation was carried out, and the results were compared with the traditional screening method based on Trypticase Sulfite Neomycin (TSN) selective medium. A high-efficiency PCR screening method, targeting C. butyricum species-specific primers, was established. The method was confirmed to have high specificity and sensitivity towards C. butyricum cut-off CFU 103. Compared with the traditional method, the screening success rate of C. butyricum strains increased from 0.61 to 81.91%. The PCR screening method could quickly and accurately detect C. butyricum in samples and dramatically improve screening efficiency.
The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.
- MeSH
- bakteriální geny MeSH
- Bifidobacterium klasifikace enzymologie MeSH
- DNA bakterií genetika MeSH
- DNA primery MeSH
- fylogeneze * MeSH
- genetické markery MeSH
- genotyp MeSH
- glutaminsynthetasa genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- Publikační typ
- časopisecké články MeSH
Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired β-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-laktamasy genetika metabolismus MeSH
- DNA primery chemická syntéza metabolismus MeSH
- Enterobacter cloacae klasifikace účinky léků enzymologie genetika MeSH
- Escherichia coli klasifikace účinky léků enzymologie genetika MeSH
- exprese genu MeSH
- fylogeneze MeSH
- gramnegativní bakteriální infekce diagnóza epidemiologie mikrobiologie veterinární MeSH
- Klebsiella pneumoniae klasifikace účinky léků enzymologie genetika MeSH
- kur domácí mikrobiologie MeSH
- lidé MeSH
- maso mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- peniciliny farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. METHODS: We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. RESULTS: All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%-90% reaction positivity rate) and in their absolute reaction yield (7.3%-137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H- and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H-) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). CONCLUSIONS: Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.
- MeSH
- analýza jednotlivých buněk MeSH
- DNA primery metabolismus MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- reprodukovatelnost výsledků MeSH
- reverzní transkriptasa metabolismus MeSH
- RNA metabolismus MeSH
- superoxiddismutasa 1 genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Melanoma is the least common form of skin tumor, but it is potentially the most dangerous and responsible for the majority of skin cancer deaths. We suggest that the skin microbiome might be changed during the progression of melanoma. The aim of this study is to compare the composition of the skin microbiota between different locations (skin and melanoma) of a MeLiM (Melanoma-bearing Libechov Minipig) pig model (exophytic melanoma). Ninety samples were used for PCR-DGGE analysis with primers specifically targeting the V3 region of the 16S rRNA gene. The profiles were used for cluster analysis by UPGMA and principal coordinate analysis PCoA and also to calculate the diversity index (Simpson index of diversity). By comparing the obtained results, we found that both bacterial composition and diversity were significantly different between the skin and melanoma microbiomes. The abundances of Fusobacterium and Trueperella genera were significantly increased in melanoma samples, suggesting a strong relationship between melanoma development and skin microbiome changes.
- MeSH
- Bacteria klasifikace izolace a purifikace MeSH
- DNA bakterií genetika MeSH
- DNA primery MeSH
- Fusobacterium genetika izolace a purifikace MeSH
- genetická variace MeSH
- kůže mikrobiologie MeSH
- melanom mikrobiologie patologie MeSH
- mikrobiota * MeSH
- miniaturní prasata MeSH
- modely nemocí na zvířatech MeSH
- prasata MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
