Study On LDR Typing And Quantification Of Human Gut Bacteria

Objective: To establish a stable, rapid and high throughput typing and quantification of human gut bacteria.Method: Fecal samples were collected and bacteria DNA were extracted, with which 16S rDNA were amplified using universal primers. The products were cloned and sequenced as standard sample of the detected bacterial. 16S rDNA sequences of three kinds of dominant bacterial in gut, Clostridium coccoides group, Bacteroides and related genera and Clostridium leptum group were obtained by GenBank, according to which LDR (ligase detection reaction) probes were designed. Clones of standard samples of the three dominant bacteria were quantified with UV absorption method and mixed by certain ratio. 16S rDNA were PCR amplified, with the products, LDR were carried out and ligation product signals were measured to establish a standard curve. The sensitivity of this method is determined as the detectable threshold concentration value of serially diluted standard sample. 50 clinical fecal samples were collected from patients of different ages and genders, from which total bacteria DNA were PCR amplified for 16S rDNA and LDR detection. The relative ratio of the three kinds of bacteria was calculated. The results were evaluated by SPSS.Results: With the designed universal primers for screening, 16S rDNA of the samples were cloned and sequenced, and a group of standard samples were obtained, with which a standard curve was successfully established. The lowest detectable level of this method is 10-3fM. Results in the 50 clinical samples showed, the relative content of Clostridium coccoides group increased with older age (p<0.01); a dramatic increase of the relative content of Bacteroides related genera was observed in aged subjects (p<0.01), but no difference was found between the young and middle-aged subjects (p>0.05); there was not any difference observed of the relative content of Clostridium leptum group among the groups, either (p>0.05). No difference was found in the relative content of three dominant gut bacterial between different genders (p>0.05). In all populations, Clostridium coccoides group has higher proportion (46.5±10.8%) than Bacteroides related genera (29.3±9.2%), Clostridium leptum group has the lowest proportion (24.2±11.5%). The biological mechanism remains to be elucidated.Conclusions: Comparing with using directly genome DNA of anaerobic bacterium, cloned and jointed plasmid PCR products would be more convenient as standard samples, since the repeatedly culture of the bacterial under strict circumstances could be avoided, which also ensures the quality and accuracy of subsequent experiments. The combination of PCR and LDR for the detection of gut bacteria could achieve the highly sensitive quantification of different kinds of bacteria at the same time, making it a simple, rapid, stale, universal and high throughput method of detection and typing. According to the results and statistical data from clinical samples, the relationships between the relative contents of three dominant gut bacterial and age, gender were primarily demonstrated, and their relative contents in different populations were also obtained. Yet the exact biological mechanism underlying the results still needs further investigation.