Design Of The Species-specific Primers For Natural Complex Microbial Community

The study of microbial community structure has been a hot research contentof microbial ecology and environmental science. In addition to the traditionalmicrobiological culture methods, more research is to analyze the samples bydirect extraction of total community DNA or RNA and quantify the levels ofdifferent species using the real-time fluorescence quantitative techniques or othermolecular biology techniques. Specific primer design plays an important role inthe process. How to design and evaluate the species-specific primers is theprimary problems to be solved.The main topic in the study mainly contains two aspects:Firstly, we developed a C++/C#software named Sequence-Analysis usingour own specific algorithm based on successive window-sliding and matching.We used it to search for short unique fragments in different16S rDNA sequencesof the GuJing Gong microbial community and to check the experimental result sthrough PCR (Polymerase Chain Reaction) amplification.Secondly, we designed130pairs of primers for species with whole genomeinformation, the species-specific marker gene and the non-16S gene. After PCRamplification and electrophoresis, we counted the amplifiction result andassessed the merits of the three primer design programs through primers’amplification success rate and primers’ specific amplification success rate.In this study, we designed130pairs of primers for GuJingGong pitsrespectively using the whole genome, the species-specific marker gene andnon-16S gene. we found that the amplification success rate of using wholegenome is36.14%, the species-specific marker gene is44.74%and theamplification success rate using the non-16S gene is33.33%. The specificamplification success rate of whole genome, specifies-specific marker gene andnon-16S gene is26.50%,31.58%and33.33%. From the results, using thespecies-specific marker gene is more efficient than using the whole genome. Inthe part of specific amplification, using specific genes is equal to the met hodusing non-16S gene. They are both higher than the method using whole genomemethod. It is recommended that primers based on species-specific genes are best forspecific amplification in the microbial community. For those species yet withoutspecies-specific genes, primers based on the whole genome sequences arerecommended. For those species yet with neither species-specific genes norwhole genome sequences, primers based on non-16S rDNA genes may be worthtesting.