PCRbased Methods

PCR has revolutionized microbial ecology, resulting in the development of several techniques of microbial community fingerprinting. Although most of these methods are generally based on the amplification of only the variable regions or the totality of the 16S rRNA genes, amplified fragments can also derive from total RNA extracted from food and amplified by reverse transcriptase-PCR (RT-PCR).

Table 1.1 Summary of the Most Widely Used Culture-independent Techniques and Their Applications to Microbial Ecology

PCR-based Methods

- PCR-DGGE/PCR-TGGE

- PCR-ARDRA

- RISA/ITS-PCR

In situ Methods

- Multiplex FISH

- Fluorescence in situ PCR

Other methods

- Flow cytometry

Taxonomic Resolution

Community members (genus/

species level) Community members (genus/ species level)

Community and population members (genus, species, strain level)

Community members (genus/

species level) Community members (species level)

Particular community members

(species groups level) Population members (strain level)

Community and population members (genus, species, and strain level)

Community members (species level)

Community members (species level)

Community members (species level)

Population members (strain level)

Applications to Microbial Ecology

Community fingerprinting;

population dynamics Mutation analysis; community fingerprinting; population dynamics Community fingerprinting; dynamics between (species-dynamics) and within (strain-dynamics) populations Community fingerprinting;

population dynamics Automated assessment of microbial diversity within communities of isolated microorganisms Community fingerprinting;

population dynamics Automated estimation of diversity (typing) within populations Automated estimation of diversity within communities (species composition) and populations (typing)

Detection of viable (both cultivable and uncultivable) cells within communities; temporal and spatial distribution of microbes within ecosystems Similar to FISH; simultaneous investigation of complex communities (e.g. biofilms) Detection of viable, slow-growing cells within communities; sensitive identification of target sequences with low copy number

Selective enumeration of mixed microbial populations and sub-populations; physiological cell state analysis.

(continued)

Table 1.1 Summary of the Most Widely Used Culture-independent Techniques and Their Applications to Microbial Ecology (continued)

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