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Abstract
Human body is one of the most complex and diverse microbial ecosystem in which various microbes are living together with their hosts. Starting with Louis Pasteur's postulation that human health is dependent on gut-resident microbiota, microbes in the gastrointestinal tract have been studied using culture-based techniques. Cultivation has the great advantage that isolates can be recovered and used to further studies for their ability to utilize different substrates and other physiological properties. However, cultivation method is very labor-intensive and can not reveal representative microbial diversity of human intestinal tract. Only small fraction of the microbes residing in human intestine can be cultured and majority of intestinal microbes (approximately 60~70% of intestinal microbes) can not be come into view with currently available cultivation techniques. To avoid reliance on cultivation, many culture-independent molecular methods have been developed to analysis environmental microbes and our understanding of complex microbial communities has been greatly increased by molecular methods in recent decades. These culture-independent methods are mainly based on the use of microbial DNA sequences. Among prokaryotic DNAs targeted by molecular analysis, approximately 1.5 kb long 16S ribosomal DNA gene that encodes part of the small subunit (SSU) of ribosome is often used for analysis of microbial diversity. Molecular techniques introduced in microbial ecology have made it possible to study the composition of intestinal flora in a culture-independent way based on the detection of SSU rDNA.
REFERENCES
1). Whitman WB., Coleman DC., Wiebe WJ. Prokaryotes: the unseen majority. Proc Natl Acad Sci U S A. 1998. 95:6578–83.
2). Amann RI., Binder BJ., Olson RJ., Chisholm SW., Devereux R., Stahl DA. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990. 56:1919–25.
3). Muyzer G., de Waal EC., Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993. 59:695–700.
4). Favier CF., de Vos WM., Akkermans AD. Development of bacterial and bifidobacterial communities in feces of newborn babies. Anaerobe. 2003. 9:219–29.
5). Namsolleck P., Thiel R., Lawson P., Holmstrom K., Rajilic M., Vaughan EE, et al. Molecular methods for the analysis of gut microbiota. Microb Ecol Health Dis. 2004. 16:71–85.
6). Franks AH., Harmsen HJ., Raangs GC., Jansen GJ., Schut F., Welling GW. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. 1998. 64:3336–45.
7). Lay C., Sutren M., Rochet V., Saunier K., Dore J., Rigottier-Gois L. Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota. Environ Microbiol. 2005. 7:933–46.
8). Zoetendal EG., Ben-Amor K., Harmsen HJ., Schut F., Akkermans AD., de Vos WM. Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes. Appl Environ Microbiol. 2002. 68:4225–32.
9). Schena M., Shalon D., Davis RW., Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995. 270:467–70.
10). Bodrossy L., Sessitsch A. Oligonucleotide microarrays in microbial diagnostics. Curr Opin Microbiol. 2004. 7:245–54.
11). Zhou J. Microarrays for bacterial detection and microbial community analysis. Curr Opin Microbiol. 2003. 6:288–94.
12). Bae JW., Park YH. Homogeneous versus heterogeneous probes for microbial ecological microarrays. Trends Biotechnol. 2006. 24:318–23.
13). Wilson KH., Wilson WJ., Radosevich JL., DeSantis TZ., Viswanathan VS., Kuczmarski TA, et al. High-density micro-array of small-subunit ribosomal DNA probes. Appl Environ Microbiol. 2002. 68:2535–41.
14). Lazarevic V., Whiteson K., Huse S., Hernandez D., Farinelli L., Osterås M, et al. Metagenomic study of the oral microbiota by Illumina high-throughput sequencing. J Microbiol Methods. 2009. 79:266–71.
15). Akhras MS., Thiyagarajan S., Villablanca AC., Davis RW., Nyren P., Pourmand N. PathogenMip assay: a multiplex pathogen detection assay. PLoS ONE. 2007. 2:e223.
16). Claesson MJ., O'Sullivan O., Wang Q., Nikkila J., Marchesi JR., Smidt H, et al. Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine. PLoS ONE. 2009. 4:e6669.
17). Quince C., Lanzen A., Curtis TP., Davenport RJ., Hall N., Head IM, et al. Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods. 2009. 6:639–41.
18). Gomez-Alvarez V., Teal TK., Schmidt TM. Systematic artifacts in metagenomes from complex microbial communities. ISME J. 2009. 3:1314–7.
19). Qin J., Li R., Raes J., Arumugam M., Burgdorf KS., Manichanh C, et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010. 464:59–65.
20). Qiu X., Wu L., Huang H., McDonel PE., Palumbo AV., Tiedje JM, et al. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl Environ Microbiol. 2001. 67:880–7.
21). Polz MF., Cavanaugh CM. Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol. 1998. 64:3724–30.
22). Huber T., Faulkner G., Hugenholtz P. Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics. 2004. 20:2317–9.
23). Jeong H., Barbe V., Lee CH., Vallenet D., Yu DS., Choi SH, et al. Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). J Mol Biol. 2009. 394:644–52.
24). Reyes A., Haynes M., Hanson N., Angly FE., Heath AC., Rohwer F, et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature. 2010. 466:334–8.
Table 1.
Notable features of molecular methods for studying the intestinal microbiota
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