| The analysis of the composition of intestinal flora has become a key research target in the field of life sciences,as it is closely related to host's health and disease.However,the high viscosity of bacteria in fecal samples and their interference with uneven distribution of impurities and solids,as well as the loss of subdominant species during DNA extraction,have seriously affected the analysis results of intestinal flora.Therefore,the aim of this study was to improve the pretreatment method for samples of intestinal microbiota and optimize the sample-separating conditions for liquid chromatography.The auxiliary effects of this method in improving the accuracy and sensitivity of separation and analysis of intestinal flora were disscussed.The research included two parts,which were the modelling system of intestinal flora and rat intestinal flora experiments.The method and results were shown as follows.1.The model strains of intestinal flora:High performance liquid chromatography(HPLC)was used to investigate the chromatographic behavior of the mixture of 9 typical intestinal microflora and single strain therein,respectively;the optimal elution method of HPLC for separation and analysis of the mixed strains was successfully established,which was the step gradient elution performed with piperazine-HCI buffer containing 0.10-0.70 mol/L NaCl.The maximum sample load of the chromatographic column was 500?L of 5-fold concentrate of the bacterial broth with an OD600 of 0.576,and the established HPLC method had high resolution and good reproducibility.Five eluted fractions were separated by HPLC and the separating effect thereof was analysized by the multiplex PCR method.The results showed that the multiplex PCR assay is able to rapidly and specifically detect bacteria in the 5 elution fractions seperately,and the amplified product sequences of the five elution fractions showed 98%-99%sequence homology to the bacteria DNA in the Genbank database;false-negative/false-positive results were amplified erroneously and showed obscure strip in electrophoresis during the detection of untreated samples.The HPLC method for the separation and analysis of the intestinal microflora provides a basis for further investigation of the composition and structural diversity of the intestinal microflora in real fecal samples.2.Rat Intestinal Microbiota:Liquid chromatography was used as the pretreatment method for fecal samples,the sample preparation conditions of the intestinal microflora were optimized,the liquid chromatography conditions were optimized and characterized,the bacterial cells were recovered and their DNA was extracted.High-throughput sequencing results indicated that the composition and abundance of the intestinal microflora of the ML-OSE group(mixation-type loading_one step elution,that is,the intestinal microflora sample was fully mixed with the chromatographic packing materials and eluted directly with a piperazine-HCl buffer containing 1.0 mol/L NaCl)was significantly higher than that of the untreated group,and the species information was more abundant and complete.In addition,the ML-OSE group had high intra-group similarities,and the results were stable and reproducible.However,the dispersion degree of the samples in the untreated group was very large and the difference was obvious.The genes of total intestinal bacteria,Bifidobacterium,Bifidobacterium adolescentis of each sample were used as internal standards to perform absolute quantitative detection of real-time fluorescence quantitative PCR.The results showed that the detection rate of total intestinal bacteria and Bifidobacterium of the ML-OSE group were increased by 1.17 and 1.16 times,respectively,compared with the untreated group;the detection value of Bifidobacterium adolescentis in the ML-OSE group was as high as 81.02%-84.38%,which was only 35.49%-44.91%in the untreated group.The above results indicated that the technique of liquid chromatography used as a pretreatment method for feces samples can greatly increase the sample throughput and uniformity of the intestinal flora,as well as the extraction quality of genomic DNA extracted from the fecal samples.High quality sequencing was also achieved to ensure the scientific reliability of the data.The method has several advantages,including stable-operation at room temperature,good accuracy,high sensitivity,and good reproducibility.Therefore,the study will contribute to the detection and analysis of real and complete intestinal microflora by the techniques of modern molecular biology and next-generation sequencing. |
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