Development And Application Of A Competitive PCR Assay For Functional Bacteria In Bioremediation

The traditional methods, which relying on culture media to do culture, isolation, identification and counting, are complicated and consume long time, and have been far from satisfying rapid, specific, simple, sensitive and low cost requirement in detecting and studying microorganisms. In recent years, the rapid and automatic microbial detection approaches developed quickly, it has been up to molecular level by now, by using nucleic acid hybridization, PCR and antigen-antibody reactions to the isolation, detection, identification and counting microorganisms. So, Microbial detection has become more sensitive, rapid, accurate and convenient.Competitive PCR was invented by Gilland in 1990. Overall, it uses the appropriate internal control template and the target nucleic acid (DNA or RNA) together to do the competitive PCR reaction. The PCR products can be separated by gel electrophoresis obviously, and then be further analyzed qualitatively or quantitatively. Competitive PCR can eliminate variability among samples and tubes, quantitative broader and detect the difference in 2 times. This method doesn't need expensive equipment and it's easy to achieve the experimental conditions. So, this thesis chooses competitive PCR to establish the rapid quantitative detection technique of functional bacteria in bioremediation in the aquatic environment.Preparation internal control template is the key of the quantitative PCR quantitative analyses; the competitive PCR will be directly affected by the success or failure of the internal control template's construction. In this thesis, target bacterial DNA was identified by enzymes digestion methods, two endonucleases were used to remove a small fragment in the middle, and connect the remaining two larger fragments, then the linked product was amplified in the same PCR condition using the same primers, the truncated and homologous fragments product was obtained. The product was purified and subcloned into pMD18-T vector. Restriction analysis of plasmid, PCR and DNA sequencing indicated that the competitive template had been inserted in chromosome of pMD18-T vector. Then integration plasmide DNA can be used as internal control template. After certain percentage of dilution, the internal control template was PCR amplified together with the same volume of target bacterial DNA in the same PCR reaction, the result indicated that there is distinctly competitive linear relationship between original target DNA and internal control DNA , the equation between the number of bacteria in original sample and the mass of the internal control template was set up, by doing various bacteria counting at the same time. The specificity of the primers designed for bacteria strains J₃ and Y and the sensitivity this method were also detected in this experiment. The PCR results showed the expected specific bands can be obtained. . This competitive PCR assay method is sensitive, which can detect low to 100. 5ng/μL of DNA and only need 2 days. It improves the detection rate and save a lot of time and reagents.After the competitive quantitative PCR assay was established, it was used to test the distribution of these two types of bacteria of mud samples colleted from 5 regions of the cage culture area of Gulf in Xiangshan Port, Zhejiang Province in the winter of 2006 and spring of 2007. Five regions are the cage culture area (fish), shellfish culture area, kelp cultivation area, the nearer cage culture control plot, , farther cage culture control plot, respectively. The results show that whether in the winter samples or in the spring samples, bacteria Y are higher one or two magnitudes than J₃ bacteria. In five regions, the numbers of two kinds bacteria in the cage culture area are the highest., there were1.47×10⁴cell/g J₃ bacteria and 4.59×10⁴cell/g Y bacteria in spring. But in the same region, bacteria J₃ in the spring samples are one magnitude higher than those in the winter samples, except farther control plot. As to bacteria Y, significant increase of the number was observed only in the kelp cultivation area, no significant increase was observed in other regions.