Quantitative Analysis Of Effects Of Probiotics On Intestinal Flora Based On QPCR Technique

The host's health is closely related to the intestinal flora.Once the intestinal flora structure is disordered,various diseases occur.Therefore,it is very important to obtain information on the structure of the intestinal microflora and the regulation of its growth,especially the change of key strains(groups)that are closely related to the health of the intestine,which are more important for elucidating the occurrence and development of intestinal diseases.Numerous studies have shown that high-quality genomic DNA is the key to ensuring the smooth development of intestinal flora,and it also lays the foundation for further molecular biology and bioinformatics research.Probiotics have the ability to improve the body's health by regulating the intestinal flora,which is a hot area of research today.In this study,L.acidophilus and human feces were used as materials,and the conditions of obtaining high yield and high quality of bacterial genomic DNA were determined by beadbeater-CTAB method.Then qPCR technology was applied to analyze changes in the specific intestinal flora(species)of diarrhea,constipation,and healthy subjects under the intervention of compound probiotics,stachyose,and MF13,in order to clarify the scientific value of these key strains in the maintenance and improvement of intestinal health.The results of DNA extraction showed that high-quality fecal microbiota DNA can be obtained with Mini-Bead-Beater-16 with a bead diameter of 0.1 mm for 3.5 minutes.In this study,quantitative analysis was performed on Bifidobacterium spp.,Bacteroides,Lactobacillus spp.and F.prausnitzi in the intestinal flora of 16 volunteers.Under compound probiotics intervention,the amount of Lactobacillus spp.in the intestinal tract of the diarrhea group increased significantly in a short period of time,from 5.2±0.97 before the intervention to 3.36±0.40 in the three weeks after the intervention,and during the intervention of F.prausnitzi,it has also been at a sustained level of growth,from 11.32±1.61 to 13.51±1.26 during three weeks of intervention,Bifidobacterium spp.and Bacteroides had only small changes;the amount of F.prausnitzi in the intestine of the healthy group showed a decreasing trend,before the intervention was 12.99±0.75,and the intervention stopped to fall to 8.19±1.65,there were no significant changes in Bifidobacterium spp.,Lactobacillus spp.,and Bacteroides.Under stachyose intervention,Bifidobacterium spp.and Lactobacillus spp.in the diarrhea and constipation groups increased along with the extension of the intervention time,and they continued to approach the level of the healthy group.Bacteroides did not show any significant changes in the three groups of subjects.Stachyose promoted intestinal F.prausnitzi in subjects with diarrhea,from 11.33±0.89 before intervention to 13±0.65 after intervention,but inhibited the growth of F.prausnitzi in the intestinal tract of constipation and healthy subjects.Under the intervention of MF13,the amounts of Lactobacillus spp.,Bifidobacterium spp.,and F.prausnitzi in the diarrhea and constipation groups continued to increase with the intervention time.The amount of Bacteroides in the diarrhea group decreased from 15.9±0.15 before intervention to 14.55±0.68 in the fifth week of intervention,whereas the constipation group showed an increasing trend from 14.69±0.41 to the intervention to the fifth week to 15.12±0.48,but the diarrhea and constipation group Bacteroides,Lactobacillus spp.,and F.prausnitzi were all close to healthy levels;The Bifidobacterium spp.in the intestinal tract of the subjects had a significant increase.The Lactobacillus spp.,Bacteroides and F.prausnitzi in the healthy group were significantly lower than the pre-intervention levels under the intervention of MF13.Two kinds of qPCR methods were compared by TaqMan probe method and SYBR Green I dye method.The results showed that the trend of each group of bacteria under the two methods was basically the same.The TaqMan probe qPCR was used to quantify F.prausnitzi and Bacteroides the results were higher than SYBR Green I qPCR.The quantitative results of Lactobacillus spp.were opposite.However,the results of quantification of Bifidobacterium spp.were not significantly different.The logarithm of the copy number of F.prausnitzi and Bacteroides was more than 8 and the logarithm of the copy number of Lactobacillus spp.was basically lower than 8,while the logarithm of Bifidobacterium spp.copy number was around 8,indicating that the TaqMan probe qPCR is more sensitive and SYBR Green I qPCR is susceptible to false positive results when the target DNA is low.In summary,all three probiotics had an effect on the intestinal flora and promote the growth of some beneficial bacteria.Taking probiotics and prebiotics can promote the reproduction of beneficial bacteria to a certain extent,but it requires long-term intervention and dependence;Intervention of MF13 can promote Bifidobacterium spp.,Bacteroides and Lactobacillus spp.in people with diarrhea and constipation to a healthy level.But long-term use of MF13 in healthy people will produce some antagonism to some beneficial bacteria.In short,through the quantitative analysis of several key strains(groups)of the intestinal tract,it was confirmed that the intervention of probiotics had a significant role in improving the intestinal key strains(groups)of people with diarrhea and constipation;and the effect of probiotics in healthy people was not significant.