Terminal restriction fragment length polymorphism as a diagnostic tool in gastrointestinal disease: a preliminary study
Abstract number: O368
Soeltan-Kaersenhout D., Panneman H., Akol H., Gierveld S., Catsburg A., van Bodegraven A., Savelkoul P.H.M.
Objective: Terminal restriction fragment length polymorphism (T-RFLP) has been utilised as a typing method to examine microbial community structure in human faecal samples. Adherence of microbes to the gastrointestinal mucosa rather than luminal presence however is a prerequisite for development of intestinal disease.
To this date, the T-RFLP method has not yet been evaluated for the possibility to obtain a rapid, broad view of the (adherent) microbial composition of the human intestine and its dynamics in diseased states. This evaluation might result in a screening tool for abnormalities in the adherent gastro-intestinal microflora by way of analysing changes in terminal restriction fragment (TR-F) profiles. This study was done to obtain evidence on the performance of the T-RFLP method.
Method: The technique involves amplification of the 16S rRNA gene with two differently labeled primers, digesting the amplicon with restriction endonucleases, and retrieving the molecular weight of the labeled terminal restriction fragments (the outer ends of the amplicon) in an automated DNA sequence apparatus. The labeling of both the forward and reverse primers might lead to a more accurate identification of species within a community profile since the presence of two peaks instead of one confirm the presence of a certain species.
Firstly, cultures of three common intestinal habitants of the human gut were subjected to T-RFLP analyses, alone and in mixtures, to determine if competition in the PCR between species has an effect on the T-RFLP profiles.
Secondly, mucosal biopsies from five colon locations per patient were gathered from 20 patients for analysis by T-RFLP. The three aforementioned species were quantified in the samples by quantative real-time PCR to determine if adequate amounts were present for detection by T-RFLP.
Results: It was shown that the T-RFLP method produces consistent, reproducible profiles. The patient samples showed little to no intrapatient varation in T-RFLP profile compared to interpatient variation.
Conclusion: For reasons of simplicity, similarity between T-RF sizes of different genera should be omitted or reduced to a minimum by way of careful restriction enzym selection. Furthermore, a database containing DNA sequences of the adherent human intestinal microbial community, combined with adequate computer software capable of performing meta-analyses of forward and reverse peak presence, is essential for obtaining rapid T-RFLP profile results.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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