PCR-RFLP
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Pro zrychlení identifikace mykobakteriálních druhů z urychlené kultivace byla zavedeny a optimalizovány molekulárně biologické metody - PCR/RFLP (polymerázová řetězová reakce s následnou restrikční analýzou naamplifikovaného produktu). Pro tyto metody využíváme amplifikaci konzervativních oblastí dna J genu a HSP 65 kDa genu mykobakteriálního genomu. V období od května 1999 do června 2001 bylo identifikováno 144 kmenů klasickými metodami a zároveň metodou PCR/RFLP. Zavedením metody PCR/RFLP byla doba identifikace zkrácena z původních 3 týdnů na 2-7 dní.
A rapid procedure for the identification of cultured Mycobacterium isolates, based on the combination of enzymatic amplificauon and restnction analysis (PCR/RFLP), was optimalized and Qy the polvmerase chain reaction (PCR) is amplified part of dna J gene res. HSP 65 kDA gene common to all mycobacteria. 144 mycobacterial strains were identified parallel by conventional bacteriological determination and by PCR/RFLP from May 1999 to June 2001. Differentiation of Mycobacteria using PCR/RFLP methods can be completed within 2-7 days.
Internalin A, encoded by the gene inlA, is the key virulence factor, which allows crossing the intestinal barrier during the initial stages of an infection. The aim of this study was to evaluate the potential of RFLP (restriction fragment length polymorphism) technique of inlA for monitoring the occurrence of potentially invasive strains of L. monocytogenes from different sources. The ability to invade the epithelial host cells of the intestinal barrier was detected in 52.3 % of the investigated strains. RFLP profiles (1and 4) associated with the production of truncated form of the internalin A and with the decreased invasiveness was detected only in serotype 1/2a and 1/2c. These RFLP profiles were found in strains isolated from food (43.6 %) and the external environment (66.7 %), but also in some human strains (42.1 %). Following the inlA polymorphism by PCR-RFLP is a fast screening technique for evaluation of alteration in pathogenic potential of L. monocytogenes.
1. Pulsed-field gel electrophoresis (PFGE) and PCR-restriction fragment length polymorphisms of the flagellin gene (fla-RFLP) were used to analyse 92 poultry and 110 human strains of Campylobacter jejuni. 2. Among poultry strains, 11 fla-RFLP and 11 PFGE subtypes were found, while human strains could be divided into 23 fla-RFLP and 32 PFGE subtypes. Altogether, 31 fla-RFLP and 32 PFGE subtypes were found. 3. The results show that individual flocks in farms are mostly infected with a single C. jejuni clone, while during subsequent colonisation their genotypes altered. fla-RFLP and PFGE profiles in poultry and humans were identical in less than 6% of cases. The results found so far confirm previous findings that chicken meat does not represent as important a source of campylobacteriosis as was previously believed. 4. The typing of Campylobacter sp. forms the basis for an evaluation of the current state and risk assessment of various Campylobacter sp. sources in relation to humans. Examination of samples with only one method is insufficient for epidemiology studies, because apparently different clones identified with one method could originate from a single clone, which could be proved with the other method.
- MeSH
- Campylobacter jejuni genetika izolace a purifikace klasifikace MeSH
- financování organizované MeSH
- jatka MeSH
- kur domácí mikrobiologie MeSH
- lidé MeSH
- polymerázová řetězová reakce veterinární MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- pulzní gelová elektroforéza veterinární MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
The strains belonging to Burkholderia cepacia complex are important opportunistic pathogens in immunocompromised patients and cause serious diseases. It is possible to obtain isolates from soil, water, plants and human samples. Taxonomy of this group is difficult. Burkholderia cepacia complex consists of seventeen genomic species and the genetic scheme is based on recA gene. Commonly, first five genomovars occurre in humans, mostly genomovars II and III, subdivision IIIA. Within this study we tested identification of first five genomovars by PCR with following melting analysis and RFLP. The experiments were targeted on eubacterial 16S rDNA and specific gene recA, which allowed identification of all five genomovars. RecA gene appeared as more suitable than 16S rDNA, which enabled direct identification of only genomovars II and V; genomovars I, III and IV were similar within 16S rDNA sequence.
Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.
- MeSH
- bakteriální geny * MeSH
- Cronobacter klasifikace genetika MeSH
- DNA bakterií MeSH
- DNA primery MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- fylogeneze MeSH
- kontaminace potravin analýza MeSH
- polymerázová řetězová reakce ekonomika metody MeSH
- polymorfismus délky restrikčních fragmentů * MeSH
- potravinářská mikrobiologie * MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
DNA genotyping is among the most common analyses currently performed in scientific research. Two high-throughput genotyping techniques are widely used - the "classic" PCR-RFLP and probe-based methods such as TaqMan® PCR assay or KASP™ genotyping. The probe-based techniques are claimed to be more accurate than PCR-RFLP; however, the evidence for this claim is sparse. We have directly compared results of genotyping of two SNPs (rs1229984 and rs17817449) obtained by the PCR-RFLP and KASP™ in 1,502 adult Caucasians. The results were identical in 97.3 % and 95.9 % cases, respectively. Discrepancies (either different results or result obtained with one but not with the other method) were addressed by confirmatory analysis using direct sequencing. The sequencing revealed that both methods can give incorrect results, but the frequency of incorrect genotyping of rs1229984 and rs17817449 was very low for both methods - 0.1 % and 0.5 %, respectively, for PCR-RFLP and 0.1 % and 0.3 %, respectively, for KASP™. These results confirm that the KASP™ technique is slightly more accurate, but it achieves slightly lower call rates than PCR-RFLP. When carefully set up, both PCR-RFLP and KASP™ could have accuracy of 99.5 % or higher.
This is the first exhaustive report on the fungal community biodiversity in hypersaline water in România. A total of 27 fungal strains (19 molds and eight yeast) have been isolated from Lopătari hypersaline water, Buzau County. Based on classical investigation, these strains have been identified as belonging to the genera Aureobasidium, Alternaria, Aspergillus, Penicillium, and Fusarium. The molecular characterization of fungal isolates at species level was performed using PCR-RFLP analysis of the 5.8S-ITS region. PCR products were digested with different combinations of endonucleases. The most frequently isolated species were Aspergillus niger (14.81% of all isolates), A. versicolor, (14.81%) and Penicillium crustosum (14.81%). In addition, ribosomal restriction patterns which exhibited profiles specific to Aureobasidium pullulans were derived, and to discriminate between Aureobasidium isolates, the elongase-encoding gene (ELO) was chosen as a genetic marker followed by digestion with endonuclease HhaI. Five yeast isolates displayed restriction patterns corresponding to Aureobasidium melanogenum (18.52%) and three isolates to Aureobasidium pullulans (11.11%). In addition, the RFLP types of Aureobasidium pullulans varieties with HhaI are clearly distinguished and could be applied to assess the intraspecific variability.
- MeSH
- Ascomycota genetika izolace a purifikace metabolismus MeSH
- Aspergillus genetika izolace a purifikace metabolismus MeSH
- biodiverzita MeSH
- DNA fungální genetika MeSH
- fylogeneze MeSH
- houby klasifikace genetika izolace a purifikace MeSH
- kvasinky genetika izolace a purifikace metabolismus MeSH
- mikrobiologie vody * MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů * MeSH
- řeky chemie mikrobiologie MeSH
- ribozomální DNA genetika MeSH
- sekvenční analýza DNA MeSH
- slané vody * MeSH
- tolerance k soli * MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Rumunsko MeSH
The tench Tinca tinca is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being transplanted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efficient phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical populations and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing (EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identification of the Western and Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable researchers and fishery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for characterization of cultured strains and in breeding experiments.
- MeSH
- Cyprinidae klasifikace genetika MeSH
- fylogeneze MeSH
- mitochondriální DNA genetika MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- rybí proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Asie MeSH
- Evropa MeSH
