| At present, the antibiotic resistance is becoming a serious problem. Antibiotic resistance exists as an inevitable natural phenomenon. And social factors and human factors could accelerate the dissemination of drug resistant bacteria. The incidence of antibiotic-resistant infections increases so rapidly that once curable diseases becoming difficult to treat. Bacterial drug resistance mainly caused by the factors from bacteria itself and the external environment, including abuse of antibiotics, appearance of activating enzyme, pumping of antibiotics, changes of drugs target site, and the spreading of the resistant bacteria.Helicobacter pylori infection is a major cause for chronic active gastritis and peptic ulcer. It is closely related with gastric cancer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma. WHO has classified Helicobacter pylori as a carcinogenic factor. Its infection rate is about 50-70% in China. Therefore,to control the anti-Helicobacter pylori infection is an important medical and social issue we are facing with. With the extensive use of antibiotics, more and more H.pylori becomes antibiotics-resistant. In a long time, the identification of drug target genes and drug metabolism genes plays an important role in the clarification of drug resistance mechanism. For example, metronidazole- resistance is caused by mutation of rdxA and frxA. Clarithromycin-resistance is associated with point mutation in 23S rRNA of the H.pylori. However, a lot of resistance genes and drug resistant mechanisms are still unknown. Therefore, it is necessary to set up new methods to screen and identify drug resistance genes.Signature-tagged mutagenesis is one kind of technologies that has been often used in screening function genes of pathogenic microorganisms. It is carried out by constructing random insertion mutants and then screening in vivo. Basicly, vectors with different tags were used to construct mutant libraries. Then, animals, cells or other models were infected. The mutant strains will die due to losing of the pathogenic capacity or ability to resistant stress from environment. The tags will thus not be detected in the recovery sample. Finally, hybridization or PCR was done to detect the tags within the recovery sample. The missing tags will indicate that the mutant genes were virulence-associated or with specific functions. High-throughput screening method is an effective way to screening virulence and resistant genes and a variety of specific function-related genes.In this study, total DNA of Helicobacter pylori was digested at random with blunt-end restriction enzym. Then the 300-500bp DNA fragments (Fr) after digestion were recovered and cloned in suicide plasmid of tags and then transferred into E.coli DH5αby electroporation. 1200 recombinant plasmids were obtained by selection culture on chloramphenicol-resistance plates. It covered a most part of genome sequence. This work lays a foundation for further large-scale construction of H.pylori mutant library and identification of drug-resistance genes of H.pylori.The tuberculosis is a kind of communicable disease and one of threatens important for human health. Among world population, 1/3 is infected with mycobacterium tuberculosis. It is a big problem of public health and social in the 21st century. Therefore, DNA fingerprinting analysis by MTB had been used to clarify clinical molecular epidemiological characters. It is much helpful for understanding the genetic diversity of MTB, and also very valuable for the foundation study as well as lemology mechanism, drug-resistance mechanism, and controling of resistant strains. DNA fingerprinting had been used in the study of clinical molecular epidemiology aready. IS6110-RFLP is the―gold standard‖for the genotyping of MTB. IS6110 was often used to detect variations in inserts sequences. rep-PCR can amplify short repeat sequences, and then were revealed diversity against the genome.In this study, total DNA of resistance mycobacterium tuberculosis was extracted and rep-PCR was completed. The DNA fragments were separated with Agilent 2100 Bioanalyzer. The data were analyzed using DiversiLab software. These strains were divided into four genotypes according cutoff of 70% similarity. It showed that there was genetic diversity between genome of strains. There is not dependablity obviously between genotype and drug resistance. It was suggested that drug resistance arose independent genotype. Type I and types II were perhaps the main epidemic strains. And the MTB control strategy should be construted all-around. |
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