Browsing tag: sekwencjonowanie

Metody genotypowe i fenotypowe wykorzystywane w typowaniu drobnoustrojów do celów epidemiologicznych

The application of genotyping and phenotyping techniques for epidemiological analysis of microorganisms
M. Brzozowski, P. Kwiatkowski, D. Kosik-Bogacka, J. Jursa-Kulesza

1. Wstęp. 2. Metody fenotypowe. 2.1. Biotypowanie. 2.2. Typowanie fagowe. 2.3. Analiza profili lekowrażliwości. 2.4. Metody analizy białek. 2.5. Spektroskopia mas. 3. Metody genotypowe. 3.1. Genotypowanie bez wykorzystania sekwencjonowania. 3.1.1. REA-PFGE. 3.1.2. RFLP i PCR-RFLP. 3.1.3. AFLP. 3.1.4. RAPD. 3.1.5. Mikromacierze (CHIP DNA). 3.1.6. MLVA. 3.2. Metody genotypowe wykorzystujące sekwencjonowanie. 3.2.1. Technologie sekwencjonowania. 3.2.2. MLST i SLST. 3.2.3. WGS – wgSNP, cgMLST, wgMLST. 3.2.4. Zalety i wady WGS. 4. Popularność metod typowania bakterii w badaniach biomedycznych na podstawie analizy bazy PubMed. 5. Podsumowanie

Abstract: The research on similarity between bacteria in outbreak investigations enables the identification of bacterial strain responsible for infections, their source and modes of transmission. These investigations are also necessary for the analysis of spreading of bacteria, not only locally, e.g. in a hospital in a specific country, but also internationally and globally. Therefore, it is of great importance to have the most up to date knowledge regarding different methods used in bacterial typing. This review discusses and compares methods facilitating bacterial typing at a strain level. Phenotyping methods analysed in this article are: Biotyping, Antimicrobial Susceptibility Typing, Phage Typing and protein-based methods. Genotyping techniques reviewed in this article are based on digestion of genomic DNA, methods using amplification of DNA, and based on sequencing DNA. This would include Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS). Methods used in identification of bacterial strains are being constantly improved, and gaining more in depth knowledge and familiarising with their effectiveness enables better analysis and control of epidemiological situation e.g. in hospitals.

1. Introduction. 2. Phenotyping methods. 2.1. Biotyping. 2.2. Phage typing. 2.3. Antimicrobial susceptibility typing. 2.4. Protein-based methods. 2.5. Mass spectrometry. 3. Genotyping methods. 3.1. Genotyping without DNA sequencing. 3.1.1. REA-PFGE. 3.1.2. RFLP and PCR-RFLP. 3.1.3. AFLP. 3.1.4. RAPD. 3.1.5. Microarrays. 3.1.6. MLVA. 3.2. Genotyping using DNA sequencing 3.2.1. Sequencing technologies. 3.2.2. MLST and SLST. 3.2.3. WGS – wgSNP, cgMLST, wgMLST. 3.2.4. Advantages and disadvantages of WGS. 4. Popularity of typing methods in biomedical research – PubMed database analysis. 5. Conclusions

Wybrane metody molekularne wykorzystywane w ocenie bioróżnorodności mikroorganizmów glebowych

Selected molecular methods used in assessing the biodiversity of soil organisms
M. Łyszcz, A. Gałązka

1. Wstęp. 2. Metody klasyczne stosowane do identyfikacji bakterii. 3. Metody molekularne stosowane do identyfikacji mikroorganizmów glebowych. 4. Analiza zawartości zasad G+C w DNA. 5. Hybrydyzacja kwasów nukleinowych. 6. Analiza sekwencji kwasów nukleinowych. 7. Podsumowanie

Abstract: Biodiversity and the identification of new important features of microorganisms is crucial for the development of biotechnology. The current knowledge about microbs in natural environments is limited, thus the analysis of the microbial diversity in nature is not an easy task. So far, only a small percentage of prokaryotic microorganisms has been identified. It is believed that the soil environment is one of the richest reservoirs of microorganisms, as approximately 2 000 to 18 000 prokaryotic genomes can be isolated from one gram of soil. In this publication the selected methods used to identify microorganisms are presented. The first molecular marker used in the genetic identification of soil microorganisms was the analysis of the G+C base content, sincemicroorganisms exhibit differences in the (G+C)/(A+T) relative factor. Another method used to identify bacteria is the nucleic acid hybridization. This technique involves a determination of the degree of similarity of DNA-DNA between two organisms. One of the most frequently used hybridization technique is FISH – fluorescent in situ hybridization. The most precise method for analyzing the nucleic acids is sequencing, i.e. determining the order of nucleotides which form the genetic information of the microorganism studied. Very often in molecular studies the 16S rDNA molecule is subjected to sequencing.

1. Introduction. 2. Classical methods used to identify bacteria. 3. Molecular methods used in the identification of soil microorganisms. 4. Analysis of the G+C DNA content. 5. Nucleic acid hybridization. 6. Analysis of nucleic acid sequences. 7. Summary