Rapid Analysis Of Intestinal Microflora In Chronic Diarrhoeal Patients And Application Study By 16S RDNA Terminal Restriction Fragment Length Polymorphism (16S RDNA T-RFLP)

The microflora in the human intestinal tract is an extremely complexcommunity, which contains more than 400 bacterial species. Thepredominant bacteria present in human colon are anaerobe. Thesemicro-organisms are interactive , maintaining the bacterial populationdynamic balance of the human intestines. The balance plays significantroles in the digestion and absorption of food, metabolize drugs and foreigncompounds, and help prevent pathogens from colonizing thegastrointestinal tract. Establishing a new method to rapidly detect andanalyse the microfolra in the human intestinal tract is needed to improvediagnostic level of chronic diarrhea and help us to use antibiotic properlyand maintain intestinal microecological balance. The traditional methodsfor identification of faecal bacteria require various culture techniques,bacteriological isolations and biochemical tests. These methods areextremely labour-intensive and time-consuming. Previous problems ofidentification because of the difficulties in growing certainmicro-organisms on culture media-many intestinal micro-organisms cannotyet be cultivated-have been overcome by detecting molecular markers oftheir presence or activity. In particular, techniques based on 16S rDNAgene have become widely accepted because of its highly conservation andrelatively stable variation. PCR based detection of 16S rDNA gene hasbeen used for direct identification of various bacteria. In order to utilizeabove gene efficiently, we choose a pair of universal primer aiming at 16SrDNA gene. Such method, along with capillary electrophoresis, is appliedto the human digestive tract. This study is aiming to simultaneouslyidentify multiple organisms in a single sample , thus allowing previouslyimpossible descriptions of complex ecosystems. This method utilizes oneprimer labeled with 6-carboxyfluorescein amino hexy(6-FAM) fluorescentdye to detect the terminal fragment of the PCR products after digestionwith restriction enzymes. The restriction fragments are analysed bycapillary electrophoresis using an antomated DNA sequencer. We keep theresult simplified greatly due to overcoming the problem of producing largenumbers of restriction fragments. The data is analysed with GeneScanAnalysis Software. Different bacteria will be analysed qualitatively andquantitavely through their different patterns rapidly. The method was usedfor the detection of intestinal microbes of 30 cases with chronic diarrheaand 40 cases of normal controls to explore the bacterial populationdynamics of the human intestines. The method was, however, found to besuitable for detecting predominant members of the intestinal microflorasimultaneously, and the predominant bacteria present in normal controlsintestines are anaerobe. The results is now different from the originalviewpoint before the metaphase in the sixties of the twentieth century,when we took E.coli for main dominant bacteria because of imperfecttechnology of anaerobe, in fact, it is minor and in a subordinate position.All patients were not found specific pathogenic bacteria , but the kind andproportion of predominant bacteria were markedly changed. It suggests thatIntestinal microflora in all patients was disturbed. So we insist thatdetection of intestinal microflora in patients with diarrhea intangibly shouldbe conducted timely to avoid abuse of antibiotic and produce ofresistant-antibiotic strains efficiently. This study proved that the use ofT-RFLP based on 16S rDNA gene is rapid, specific, sensitive and capableof detecting anaerobe difficult to cultivate and identifying multipleorganisms in a single sample with a universal primer. This technology is ofgreat utility for determing the clinical diagnosis and therapy.