The Study Of The Flourecent Quantitative PCR Method In Detection Of Bacteria Fragilis

Objective:Bacteria fragilis(B.f) is a kind of anaerobic, gram-negative non-spore bacillus, as a kind of important opportunistic pathogens,many infection disease are caused by it. Bacteria fragilis is also a predominant species in human intestinal microfloa,which plays an important role in maintaining the stability of intestinal microorganism.The classical culture methods of bacteriode species is labor-intensive and time-consuming, Moreover,classification and identification based on biochemical test do not always provide clear-cut results .The conventional quantification methods of Bacteria fragilis is unreliable,and it can not show the real change of microflora. so it is necessary to set up a quick and reliable method to detect Bacteria fragilis.Recently, great attention have been attracted on fluorescent quantitive PCR(F-Q PCR)based on 16S rRNA sequence as a reliable method for detection and identification method, and it has show great potential in the detection of bacteria. The elementary study on the detection of Bacteria fragilis by F-Q PCR based on 16S rDNA was carried out on Bacteria fragilis in the experiment.Method:A pair of primers were designed,the front primer was from the number of the gene sequence 831~853 nt,and the reverse primer was from 1005~987 nt in 16S rRNA of Bacteria fragilis. The standard curve was made by 10 times geometric proportion serial dilution of both Bacteria fragilis ATCC 25285 DNA and target gene.16S rRNA based F-Q PCR was carried out on all 22 strains and two kind of standard samples, the Bacteria fragilis was detected by F-Q PCR, the detection results were compared with that by biochemical method, the quantity of the bacteria fragilis were measured by using this standard curve, the quantification results by standard curve was compared...