Investigation Of Gastrointestinal Microbial Flora In Piglets With Different Ages By PCR-DGGE

There are a great variety of species and huge number of microorganisms living in the body of animals. The normal flora is a necessary condition for survival of animals, its diversity and change of structure was important in studying on gastrointestinal microbial community and was closely correlative with health and the development of disease. Denaturing Gradient Gel Electrophoresis (DGGE) accord to the different melting temperature carries on the separation accord to specificity melting-out temperature of the sequence which amplified by PCR of 16SrDNA variation area, and may examine difference of one nuclear acid in the sequence. Therefore through this technology amplifying the variation area of the 16SrDNA directly, constructing finger printing of DNA and establishing the fast platform of type of molecular,can widely use in the intestinal tract complex microorganism area is the bacteria colony structure succession and the multiple analysis research, and this is a very useful and widely used molecular biology way to study the diversity and changes of construction of gastrointestinal microbial community.In order to detect intestinal microbial community diversity and changes of duodenum,ejunum,ileum,caecum,rectum of piglets at 0 day,1 day,3 day,9 day,15 day, 21 day,28 day and 35 day,an PCR-DGGE for analyzing the construction of intestinal microbial community in the piglet intestinal tract was developed, and the construction of intestinal microbial flora in every part of intestine of normal piglets at different ages was detected in this study.In order to lay the Practical fundament for the research of gastrointestinal microbial flora in piglets. The articIe established a DGGE detecting Plan to research the bacteria varieties in the laboratory. Firstly, in order to understand The variations disciPline,find the dominant flora and the bacterial diversity of gastrointestinal microbial flora in piglets is ana1yzed by PCR-DGGE; Secondly,the same and special bands are recovered cloned and sequenced,through comPared with the known Sequences in Genbank database by use of Blast tools,the highest homology with the DNA sequences are identified,and at last Sequences are ana1yzed.Using DGGE method to analyze the bacterial diversity,DGGE bands were separated obviously by the gel concentration of 7.15%,30%-60% denaturant gradient in the range between 60℃temperature and 85V electroPhoresis about12h . The result indicated that little number of bacteria was found in intestinal tract even in the newly postnatal piglets , and the number and species of bacteria in every part of intestines were increasing with the growing of piglets. The number and species of of bacteria in piglet intestines at 3 day were very rich. The increasing of number of bands of the piglets was torpidity when the piglets were 9 and 15 days old,the number and species of bacteria reached peak when the piglets were 20 days old, Theincreasing of number of bands of the piglets was torpidity when the piglets were 28 days old, the number and species of bacteria reached the maximum peak when the piglets were 35 days old.of all parts, the number and species of bacteria in duodenum and jejunum were relatively less , but those of bacteria in caecum and colon were the maximum.DGGE patterns of total, as well as some special bands for sequencing were as follows: gel-cut strip of the bacteria represented in the database with the existing bacteria have very high homology, the majority of 90% or more, homology even reached 100%. A0-1 bands with clostridium perfringens has been identified as a 100% similarity, indicating that piglets born a few hours clostridium perfringens has been found in piglet intestinal paragraphs. A1-1 and Escherichia coLi identified 99% similarity, indicating that after a day of piglets born in Escherichia coLi intestinal sections has become the dominant microorganisms. In addition, the three-day-old piglets to detect intestinal LactobaciLLus acidophiLus and LactobaciLLus vaginaLis. With the increase in day-old piglet, can detect the vast majority of bacteria are not cultured. Late in the post-weaning growth process, the bacteria can not be cultured, without identification of the flora and some unknown flora play an important role.In the future researh,it is necessary to do the clone test to all DGGE bands, and using diferent molecuIar biology methods to measure the varieties of the gastrointestinal microbial community. only in this way can the states of the bacteria in the gastrointestinal system recognize more roundly and fully In addition,in the production process,we can increase the body weight and resistance of piglets through detecting and controlling the dominant flora,then a theoretical basis is provided to Piglet probiotics for the research, development and application.