Single Nucleotide Polymorphism-based QPCR Technology For Rapid Quantification Of Microbial Community Composition

Complex microbial community structure analysis and dynamic monito ring have always been hot problems of microbial ecology. However, the technology that can analyze total community structures efficiently, inexpensively and rapidly is not well developed because of abundance and complexity of organisms in almost all natural microbial community. The 16 S r DNA sequences in natual communities are extremely similar. The difficulty of the 16 S r DNA-based technology to quantitative the composition of a microbial community is that detecting and quantifying some similar 16 S r DNA sequences in a mixture sample rapidly in two hours is still a problem worldwide.In this study, we found that some SNPs(Single Nucleotide Polymorphisms) are at different taxonomic levels(kingdom, phylum, class, order, family, genus and species) commonly. We used the taxon-specific SNPs to design group-specific primers. Then we can use these primers to target a particular group DNA squences specificly from metagenomes obtained from environmental samples to quantitfy the abundance of the target taxon rapidly and monitor its compositional dynamics by q PCR(real-time fluorescent quantitative PCR). This project mainly includes three aspects. Firstly, we evaluated the strategy named SAP(Simple Allele-discriminating PCR) on how to introduce one additional mismatch at the adjacent site from the SNP. Secondly, the software used for assisting the SAP primers design process has been written. In the third part, we quantified the microbial community structure in Gu Jing Gong pit mud by phylum-specific primers.The innovation points of this study have three parts,(1) to design primers which can detect single-base differences from a mixture of DNA squences by introducing one additional mismatch at the adjacent site from the SNP to maximise their specificity, analysis base mismatch introduction systematically, and partic- ularly to detect the specific recognition ability of mismatch primers which have an additional mismatch at the second or third from bottom of the primers 3 ’end,(2) to use the SNPs commonly existing at different taxonomic levels in combination with SAP to investigate the microbial community structure and monitor their changes dynamicly at taxonomic levels,(3) to perform a software to make the SAP primers design process fast and general for all different environmental samples.