Browsing by Subject "PCR-DGGE"

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  • Hyytiäinen, Tiina (Helsingfors universitet, 2011)
    The human gastrointestinal tract (GIT) contains a complex microbiota which starts to develop after birth. Various factors such as age, health, diet and medication affect the composition of the GIT microbiota. The number and types of bacteria are different in each part of the GIT, but most of the bacteria are anaerobic. In faeces the number of bacterial cells is as high as 1011-1012 cfu/ml. The normal intestinal microbiota is essential for intestinal development, protein and carbohydrate metabolisms, and protection against pathogens. Sulphate-reducing bacteria (SRB) are typically anaerobic bacteria which use sulphate as a terminal electron acceptor to produce sulphide in their metabolism. Sulphate-reducing bacteria are widespread in all ecosystems including fresh water and marine sediments but are also present in the GIT. Most of SRB species are Gram-negative and they can use more than hundred compounds as electron donors. Dissimilatory sulphite reduction (dsrAB) gene is essential in sulphate reduction. dsrAB-gene encodes the enzyme called dissimilatory sulphite reductase, which is a key enzyme in the reduction of sulphite to sulphide. Recent findings suggest that SRB may have a role in human diseases, e.g. in periodontitis and inflammatory bowel disease (IBD). Connection between these disorders and SRB may be due to the highly toxic hydrogen sulphide. The aim of this study was to develop PCR-DGGE and qPCR methods for monitoring of sulphate-reducing bacteria from human faecal microbiota. In this study we used dsrAB-gene specific primers, which were used successfully in previous environmental microbiology studies. Previously published dsrAB-specific primers were used for PCR-DGGE. However, besides positive controls, two negative controls also amplified regardless of the modifications on temperature, amplification times, primers and MgCl2 concentration. In qPCR, specific and sensitive amplification was attained by using dsrA-gene specific primers. When the samples from paediatric patients with IBD (Crohn’s disease and ulcerative colitis) and healthy children were amplified, no differences were found between different disease groups. However there was a statistically significant difference (P <0.05) between the paediatric patients with Crohn’s disease who were on remission and those patients who’s disease was active (number of SRB; active<remission).