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a N, R, W, and Y are the International Union of Pure and Applied Chemistry codes for ambiguous bases.

a N, R, W, and Y are the International Union of Pure and Applied Chemistry codes for ambiguous bases.

and diet in the human populations studied. Two large studies, where an extensive array of oligonucleotide probes that targeted the major bacterial groups in the Gl-tract of northern European adults was used, showed that 62-75% of the fecal bacteria could be detected and identified. The remainder (z 30%) could either belong to members of the Archaea, Eukarya or most likely to yet unknown bacteria (90,91). These types of studies provide a valuable basis in order to eventually determine factors that change the microbiota such as lifestyle, diet or illness. Interestingly, FISH-FCM analysis of fecal microbiota of patients with UC revealed substantial temporal variations in the major bacterial groups studied (i.e., Bacteroides, C. coccoides-E. rectale, Atopobium, bifidobacteria and lactobacilli), which was further was supported by PCR-DGGE profiles (40).

NEW MOLECULAR DIVERSITY APPROACHES Real Time PCR

Real time quantitative PCR (qPCR) of the 16S rRNA gene is being developed the last few years for the detection and quantification of human intestinal microbiota, which has the advantages of being high throughput and measuring from 1 to up to 108 CFU (99). Both SYBR Green I and TaqMan chemistries have been used to target Bacteroides fragilis, Bifidobacterium species, E. coli, L. acidophilus and Ruminococcus productus, and the method was demonstrated to be easier and faster than dot-blot hybridization methodology (100). Real-time qPCR (50 nuclease PCR assay) has been used to study the microbiota that adhere to the colonic mucosa (101). The primer-probe combinations were applied to DNA for the detection of E. coli and Bacteroides vulgatus from pure cultures and colonic biopsy specimens. The assay was very sensitive detecting as little as 1 and 9 CFU of E. coli and B. vulgatus, respectively. Many of the qPCR assays being developed target the lactobacilli and Bifidobacterium species that may be incorporated in functional foods (102,103). Besides real time PCR of the 16S rRNA gene, the option to use the transaldolase gene of Bifidobacterium species has also been investigated and appeared to be superior to the former in quantifying bifidobacterial populations in infants (104). The qRT-PCR assays have been used for various applications such as comparison of healthy persons versus patients suffering from IBS (105), and in patients with active IBD (26). Recently, a TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the microbiota was developed (106). This method involved a pair of conserved primers, as well as universal and specific quantification probes, for species, group or genus in question, in a single reaction, and allowed relative and absolute quantification of bacteria in human biopsy and fecal samples. Further developments in real-time qPCR will facilitate our insight into the dynamics of the microbiota.

Diagnostic DNA Microarrays

The development of DNA oligonucleotide microarrays offer a fast, high throughput option for detection and estimation of the diversity of microbes in a complex ecosystem (107). Alternative terms for the microarrays are phylochips, microbial diagnostic microarrays and identification arrays. Their principle is based on the dot-blot hybridization described above. Typically microarrays contain hundreds of oligonucleotide probes, usually based on the 16S rRNA gene, specific for different strains or species or genera of microorganisms that are detected in a single assay. Total DNA or RNA is isolated from the sample, fragmented, and amplified by PCR with the simultaneous incorporation of labeled nucleotides, or directly chemically labeled. The labeled fragments are hybridized to the probes immobilized on a surface, and following washing hybridized fragments are detected by a fluorescence scanner. There are many different forms of arrays to which the probes can be attached including macroarrays, and glass microarrays that are low to medium density, and very high density Affymetric microarrays (> 104 probes typically 25 mer per chip) (108). Three-dimensional form microarrays such as the Pamgene system and gel-pads allow the option for quantitative detection (109). Studies are underway to apply microarray technology to the human intestinal microbiota (16). A macroarray membrane-based method with 60 40-mer oligonucleotide probes specific for the dominant microbiota demonstrated the feasibility of arrays for detection (110). The high throughput potential of arrays will undoubtedly encourage further efforts in this area in the coming years.

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