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
- Bacteriological Techniques economics methods MeSH
- Staining and Labeling methods MeSH
- Time Factors MeSH
- Digoxigenin metabolism MeSH
- Escherichia coli genetics isolation & purification MeSH
- Virulence Factors genetics MeSH
- Nucleic Acid Hybridization methods MeSH
- Escherichia coli Infections microbiology veterinary MeSH
- Chickens MeSH
- Humans MeSH
- Membrane Proteins genetics MeSH
- Oligonucleotide Probes genetics MeSH
- Carrier State microbiology veterinary MeSH
- Escherichia coli Proteins genetics MeSH
- Sensitivity and Specificity MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study 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 analogs & derivatives MeSH
- DNA, Bacterial genetics MeSH
- Research Support as Topic MeSH
- Hybridization, Genetic genetics immunology MeSH
- Drug Resistance genetics immunology MeSH
- Lincomycin analogs & derivatives analysis chemistry MeSH
- Chromosome Mapping methods MeSH
- Open Reading Frames genetics MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Gene Expression Regulation, Bacterial MeSH
- Blotting, Southern methods utilization MeSH
- Streptomyces coelicolor enzymology genetics MeSH
- Streptomyces enzymology genetics MeSH
- MeSH
- Digoxigenin diagnostic use genetics MeSH
- Erythrocyte Membrane pathology MeSH
- Erythrocytes enzymology pathology MeSH
- Genetic Therapy methods MeSH
- Gene Conversion methods MeSH
- Humans MeSH
- Oligonucleotide Probes immunology MeSH
- Thalassemia diagnosis genetics therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
