In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.
The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10(3) CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.
- MeSH
- bakteriologické techniky ekonomika metody MeSH
- barvení a značení metody MeSH
- časové faktory MeSH
- digoxigenin metabolismus MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- faktory virulence genetika MeSH
- hybridizace nukleových kyselin metody MeSH
- infekce vyvolané Escherichia coli mikrobiologie veterinární MeSH
- kur domácí MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- oligonukleotidové sondy genetika MeSH
- přenašečství mikrobiologie veterinární MeSH
- proteiny z Escherichia coli genetika MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
The first insight into celesticetin biosynthetic gene cluster of S. caelestis is presented. The genomic DNA of producing strain was digested, digoxigenin-labeled and hybridized with a set of probes designed according to S. lincolnensis gene sequences. Genes with high homology to the lincomycin biosynthetic genes coding for the predicted common parts of the pathway were identified in S. caelestis. Then, genomic DNA of S. caelestis treated by a multiple digestion was hybridized with five digoxigenin-labeled probes to construct a rough restriction map. Two consecutive islands formed by the genes with a putative function in biosynthesis of the shared saccharide moiety revealed an organization similar to the lincomycin biosynthetic gene cluster. The celesticetin cluster was mapped and essential information was obtained for subsequent steps, i.e. isolation and sequence analysis of the cluster.
- MeSH
- digoxigenin analogy a deriváty MeSH
- DNA bakterií genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- hybridizace genetická genetika imunologie MeSH
- léková rezistence genetika imunologie MeSH
- linkomycin analogy a deriváty analýza chemie MeSH
- mapování chromozomů metody MeSH
- otevřené čtecí rámce genetika MeSH
- polymerázová řetězová reakce metody využití MeSH
- regulace genové exprese u bakterií MeSH
- Southernův blotting metody využití MeSH
- Streptomyces coelicolor enzymologie genetika MeSH
- Streptomyces enzymologie genetika MeSH
- MeSH
- digoxigenin diagnostické užití genetika MeSH
- erytrocytární membrána patologie MeSH
- erytrocyty enzymologie patologie MeSH
- genetická terapie metody MeSH
- genová konverze metody MeSH
- lidé MeSH
- oligonukleotidové sondy imunologie MeSH
- talasemie diagnóza genetika terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
