Study On The Diversity And Changes Of Bifidobacterium In The Intestinal Tract Of Infants

The colonization of Bifidobacterium in infant intestine is very important for the healthy growth of infant.With the deepening of the research,more and more evidence shows that many factors,such as birth mode,feeding mode and the use of antibiotics,have a significant impact on the establishment of intestinal flora in infants.As the dominant species in the intestinal tract of infants,the content and diversity of Bifidobacterium have always been the focus of research.However,conventional techniques currently in use tend to ignore Bifidobacterium with low intestinal abundance.In order to study the change rule of Bifidobacterium in intestinal tract of infants,this study mainly did the following aspects:(1)comparing the whole genome sequences of 57 model Bifidobacterium strains in NCBI database,selecting their common and single copy core genes as target fragments,designing Bifidobacterium genus-specific primers,and screening specific primers through primary screening,re-screening and verification test;(2)absolute quantitative analysis of Bifidobacterium in feces of 33 newborn infants was carried out by ddPCR technique,and the effects of region,birth mode,feeding mode and ethnic group on Bifidobacterium in intestinal tract of newborn infants were discussed.(3)ddPCR and SMRT sequencing techniques were used to determine the content and diversity of Bifidobacterium in fecal samples of infants aged 5 days,15 days,30 days,60 days,90 days,120 days,150 days and 180 days in the same area,and the changes of diversity of Bifidobacterium in intestinal tract of infants in different periods were revealed.The results are as follows:1.In this experiment,a pair of Bifidobacterium genus-specific primers Bif-D-9,could amplify Bifidobacterium but not non-Bifidobacterium(20 strains),and the lower limit of quantitative detection of this primer was0.2×10-2 copies/uL.It showed good specificity and high sensitivity.2.It was found that the detection rate of Bifidobacterium in 33 infant feces was 97%(32/33),and the average content was 1.9×106 copies/g(1.0×103-2.8×107 copies/g).Further analysis found that the place of birth affected the content of Bifidobacterium longum and Bifidobacterium animalis intestine,the mode of delivery affected the content of Bifidobacterium angulatum and Bifidobacterium thennophilum in intestinal tract of infant,the way of feeding affected the content of Bifidobacterium pseudocatenulatum in intestinal tract of infant,ethnic factors affected the content of Bifidobacterium longum and Bifidobacterium pseudocatenulatum in intestinal tract of infant,It shows that individual genetic differences,birth patterns and other factors will affect the content of Bifidobacterium in the intestinal tract of infants.3.The data of ddPCR showed that the content of Bifidobacterium in the intestinal tract of infants changed dynamically.The content of Bifidobacterium increased gradually after 30 days of age,reached the peak at 90-120 days of age,and then began to decrease.According to the analysis of SMRT results,it was found that in the composition of Bifidobacterium dentium(37%),Bifidobacterium catenulatum(32%),Bifidobacterium breve(22%)and Bifidobacterium animalis(8%).Bifidobacterium breve increases with the increase of infant age,while Bifidobacterium animalis decreased gradually.In the clustering method,28 samples did not cluster by age,but by infant individuals,which showed that individual differences had a great influence on the colonization of Bifidobacterium in infant intestines.To sum up,Bifidobacterium genus-specific primer Bif-D-9 has good specificity,and is suitable for quantitative analysis of Bifidobacterium in feces and other complex samples;there are differences in the content of Bifidobacterium in the intestinal tract of infants due to the birth location,birth mode,feeding mode and ethnic factors;individual differences have great influence on the diversity of Bifidobacterium in the intestinal tract of infants.This study provides a rapid and accurate absolute quantitative Bifidobacterium genus-specific primers to analyze the changes of Bifidobacterium in infant intestines,which provides basic data and guidance for a comprehensive understanding of the composition and succession of Bifidobacterium in infants’ intestines.