Molecular methods to measure intestinal bacteria: a review

The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characte...

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Bibliographic Details
Published in:Journal of AOAC International 2012-01, Vol.95 (1), p.5-23
Main Authors: Inglis, G Douglas, Thomas, Matthew C, Thomas, Dallas K, Kalmokoff, Martin L, Brooks, Stephen P J, Selinger, L Brent
Format: Article
Language:English
Subjects:
Animals
Bacteria - chemistry
Bacteria - genetics
Chemical properties
Data Interpretation, Statistical
Denaturing Gradient Gel Electrophoresis
DNA Fingerprinting
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
DNA, Bacterial - isolation & purification
Feces - microbiology
Gene Library
Humans
Identification and classification
In Situ Hybridization
Intestines - microbiology
Microbiological chemistry
Microbiota (Symbiotic organisms)
Molecular biology
Polymerase Chain Reaction - methods
Polymerase Chain Reaction - standards
Polymorphism, Restriction Fragment Length
Research Design
Sequence Analysis, DNA - methods
Specimen Handling
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Summary:The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.
ISSN:1060-3271
1944-7922
DOI:10.5740/jaoacint.SGE_Inglis