The Research On Polymorphism Of Gastrointestinal Fungal Community In Ascites Of Patients With Cirrhosis By Culture-independent Method And ERIC-PCR

【Objective】In the past years,in the field of microecology,people have did little deep work to the fundemental problems such as diversification and ecological function of gastrointestinal fungus flora composition,however,there is a constantly increase of incidence and mortality rate due to the imbalance of gastrointestinal bacterial communities(flora)in clinic in recent years.At present,most of the laboratory around the world do their research based on the positive result of excremental culture,but we have to admit that some of the fungus can not grow on any culture medium,and thus the negative culture result can not be used to characterize strain it belongs to,so this defect restrain its application scope.For the reason mentioned above,directly extract total fungal DNA from feces sample could be a alternative strategy to comprehensively reflect the intestinal microflora.in this study we choose the QIAamp^?DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces,and then we use universal primer 18S rDNA PCR to detect fungi DNA,meanwhile,random cloning and sequence analysis was also demonstrated the quality of fungal DNA extracts using the above method.And the result of this method is also compared with the fungal isolation and culture identification methods Futher more,on the basis of the above, establishment of phylogenetic trees by cloning and sequencing and ERIC-PCR fingerprint map were utilized to analyze the polymorphism of fungal community in ascites of patients with cirrhosis.【material and method】1.Collecting stool specimens of 28 Cirrhotic Patients with Ascites,20 Chronic hepatitis B patients(non-active),30 healthy subjects.One portion was identified to genus by the fungal isolation and culture identification method,the other portion stored in-80℃within 1 hour.2.Choosing the QIAamp^?DNA Stool Mini Kit combined with bead-beating,a widely confirmed method for extracting the total DNA from feces stored in 80℃,and then we use universal primer 18S rDNA PCR to detect fungi DNA,Randomly selected 23 clones from 8 ascites of patients with cirrhosis to establish of phylogenetic trees,and the result of this method is also compared with the fungal isolation and culture identification method.3.Using ERIC-PCR fingerprint technique to analysis and compare the diversity of fungal flora in the 9 ascites of patients with cirrhosis, 8 Chronic hepatitis B patients(non-active)and 8 healthy subjects.【Result】1.Compaired with the fungal isolation and culture identification method,universal primer 18S rDNA PCR combined with QIA+bead beating(universal primer PCR detection method)is more fastly and sensitively to detect fungal colonization and infection of digestive tract,meanwhile,both of them showed that the detective rate of stool fungi from Ascites of Patients with Cirrhosis is higher than Chronic hepatitis B patients(non-active)and healthy subjects.2.In 8 ascites of patients with cirrhosis's stool specimens,there were 3 candida spp.found by common fungi culture method.but by establishing phylogenetic trees,there were found 5 candida spp.,one Aspergiilus spp.,one Saccharomyces cerevisiae and one Mortierella spp.3.ERIC-PCR fingerprint technique suggested that:the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was more abundant,compared with the Chronic hepatitis B patients(non-active)and healthy subjects.【Conclusions】1.Universal primer PCR detection method could extract high quality fungal total DNA from feces,which is beneficial to study on the diversification of gastrointestinal fungus flora composition.2.the polymorphism of gastrointestinal fungal community in ascites of Patients with Cirrhosis was relatively rich,and Candida spp.is the dominant fungi.