Construction And Application Of The Single-double Stranded Complex In Gut Microbiota Analysis

Tea is believed to have the benefits of help preventing from overweight and obesity.Recent studies have shown that there is a close relationship between gut microbiota and obesity.Polymerase chain reaction denaturing gradient gel electrophoresis(PCR-DGGE)technology has been widely applied in gut micro-ecology analysis.However,due to the length limitaion of DNA fragments,bacterial identification based on this method is not accurate enough.In order to improve the accuracy of identification,a novel DNA molecule named single-double stranded complex(SDSC)was proposed and applied in the PCR-DGGE analysis of gut microbiota in this study.With the high-fat diet(HF)and tea infusion(including Green tea,Black tea and Wuyi Rock tea)treated mice as subjects,the Bacteroides and Lactobacillus community in mice intestinal were analyzed by PCR-DGGE with SDSC approach.The main contents of this study were as follows:Firstly,a PCR-DGGE procedure for the detection of the bacteria V3 region of 16S ribosomal DNA gene was performed to assess the intestinal flora community.We found that Lactobacillus.Desulfovibrio,Moraxella,Bifidobacterium,Coiynebacterium and Enterorhabdus were the predominant bacteria in intestine of mice.The high-fat diet feeding significantly decreased 7 genus including Lactobacillus delbrueckii,resulted in the reduction of gut microbiota abundance.Whereas,green tea drinking inhibited the decrease of the relative abundance of Martelella and Anaeroplasma bacioclasticu,which induced by HF feeding,and resulted in a remarkable increase of gut microbiota richness.Both Black tea and Wuyi Rock tea inhibited the growth of Firmicutes.Secondly,a Bacteroides specific SDSC was successfully constructed and applied in DGGE analysis of the changes of Bacteroides at species level.10 bands of PCR-DGGE profiles were obtained,of which 8 bands were successfully identified as a certain species of Bacteroides by blasting from NCBI database.While,by using DGGE approach with 16S rDNA V3 region of Bacteroides,14 bands were obtained and only 4 bands could be identified.Thus,the SDSC-Bac improved the accuracy of the band identification.But the SDSC strategy may cause band lose.Finally,a Lactobacillus specific SDSC was successfully constructed and applied in DGGE to analyze the Lactobacillus changes at species level.14 bands of PCCR-DGGE profiles wereobtained,of which 9 bands were identified as a certain species of Lactobacillus by blasting from NCBI database.While by using DGGE approach with 16S rDNA V3 region of Lactobacillus,only 3 out of the 12 bands obtained could be identified as certain species.Thus,the SDSC improved the accuracy of band identification without band lose.In summary,the use of genus specific primers for SDSC construction in DGGE analysis will increase the accuracy and efficiency in bacteria indentification.However,as using longer DNA fragments,SDSC may induce band lose in DGGE analysis.This shortage may be improved by the optimization of SDSC preparation and DGGE conditions.