The Establishment And Applications Of The Methods To Quantify And Profile Feacalibacterium Prausnitzii In Human Gut Microbiota

Gut microbiota are the most complex symbiotic system and in health or disease, gut microbial variations would significantly modulate host physiology and pathology.Feacalibacterium prausnitzii is one of the predominant members in human gut microbiota which takes up more than 7% in total bacteria and spreads widely in human beings. F. prausnitzii has key physiological functions: provides energy source for intestinal epithelial cells; influences many host metabolic pathways; may has the correlation with the insulin resistance through the metabolism of the methylamine substances; has the characteristic of anti-inflammation. However, F. prausnitzii is a strict anaerobic bacterium and hard to culture. So using molecular methods to analyze the quantity and the composition of F. prausnitzii in human gut microbiota is of great significance for understanding the relationship between F. prausnitzii and human health and disease.In the first part of this study, we compared the specificity of three 16S rRNA gene-based PCR primer pairs (FPR-1-FPR-2, FPR-2F-Fprau645R and Fprau223F-Fprau420R), which are used to specifically detect and quantify F. prausnitzii. We chose the best primer pair for analyzing the quantity and composition of F. prausnitzii in further study. Clustal X was used to align the sequences of individual primer and the 16S rRNA gene of F. prausnitzii and other bacteria. The number of Faecalibacterium spp. sequences in Ribosomal Database Project (RDP) matched by each primer was obtained by the Probe Match tool. With the full-length 16S rRNA gene clone library constructed by our laboratory which contained 7255 clones from the gut microbiota of 7 Chinese people, the Simulated PCR (SPCR) program was applied to predict the clone number of F. prausnitzii and other gut bacteria matched by every primer pair; PCR amplification was performed with three primer pairs and representative clones to verified the SPCR prediction. The results showed that the first base at the 3'end of Fprau645R showed the highest mismatch level for non- F. prausnitzii bacteria. The percentage of the number of Faecalibacterium spp. sequences matched by Fprau645R accounting for that of matched bacteria sequences in the RDP database was 97.6%, which was significant higher than that of other primers. As SPCR predicted, all three primer pairs can detect about 1171 clones from F. prausnitzii; the clones of non-Faecalibacterium spp. detected by FPR-2F-Fprau645R mainly were Subdoligranulum spp., but the non-Faecalibacterium spp. clones detected by FPR-1-FPR-2 and Fprau223F-Fprau420R were mainlySubdoligranulum spp., Oscillibacter spp., Ruminococcus spp. and unclassified Ruminococcaceae etc. The real PCR showed the same results with SPCR. Therefore, the three primer pairs can detect F. prausnitzii and Subdoligranulum spp., however, the specificity of FPR-2F-Fprau645R is better than FPR-1-FPR-2 and Fprau223F-Fprau420R.Then we used the primer FPR-2F-Fprau645R to study F. prausnitzii group specific real-time quantitative PCR method and established F. prausnitzii group specific denaturing gradient gel electrophoresis (DGGE) method. And we applied these two methods to investigate the quantity and composition of F. prausnitzii in 56 obese adults and 57 normal weight adults (whom are 1:1 match in gender, age and geographical origin, female: male=63:50). The real-time PCR results showed that the number of F. prausnitzii in female gut microbiota was significant higher than that in male's (P=0.005). In the normal weight group, we got the same result as above, but in the obese group, there was no significant difference between the two gender groups. This indicated that there existed the quantitative difference in different gender groups. The number of F. prausnitzii in human feces had correlation with the body weight index (BMI) (R=0.214, P=0.023), which is probably relative to obesity. Compared with normal weight controls, there existed more F. prausnitzii in the feces of obese group, but the difference was not significant (P=0.101). Finally, we used newly built F. prausnitzii group specific PCR-DGGE method to investigate 14 obese individuals and 13 normal weight controls. There were no obvious differences in the composition of F. prausnitzii between these two groups.In summary, in this study, we used many primer specific comparision methods to select the best FPR-2F-Fprau645R among three pairs. And we analyzed the number of F. prausnitzii in human feces was significant different between two gender groups. F. prausnitzii has certain correlation with obesity; we also investigated the composition of F. prausnitzii group between obese and normal weight controls, exhibiting no obvious differences between the two groups.