title: Molecular methods to measure intestinal bacteria: a review.
abstract: 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.
authors.affiliation: Agriculture and Agri-Food Canada, 5403-1st Ave S, Lethbridge, AB T1J 4B1, Canada. Douglas.Inglis@agr.gc.ca
authors.forename: G Douglas
authors.initials: GD
authors.lastname: Inglis
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Thomas
authors.forename: Dallas K
authors.initials: DK
authors.lastname: Thomas
authors.forename: Martin L
authors.initials: ML
authors.lastname: Kalmokoff
authors.forename: Stephen P J
authors.initials: SP
authors.lastname: Brooks
authors.forename: L Brent
authors.initials: LB
authors.lastname: Selinger
mesh_terms: D000818:Animals; D001419:Bacteria / Q000737:chemistry* / Q000235:genetics; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003627:Data Interpretation, Statistical; D058645:Denaturing Gradient Gel Electrophoresis; D005243:Feces / Q000382:microbiology; D015723:Gene Library; D006801:Humans; D017403:In Situ Hybridization; D007422:Intestines / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012150:Polymorphism, Restriction Fragment Length; D012107:Research Design; D017422:Sequence Analysis, DNA / Q000379:methods; D013048:Specimen Handling
chemical_list: D004269:DNA, Bacterial