title: A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.
abstract: Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: João Carlos
authors.initials: JC
authors.lastname: Gomes-Neto
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Mantz
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Kyler
authors.initials: K
authors.lastname: Held
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Rohita
authors.initials: R
authors.lastname: Sinha
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Rafael R
authors.initials: RR
authors.lastname: Segura Munoz
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Schmaltz
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Benson
authors.affiliation: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Jens
authors.initials: J
authors.lastname: Walter
authors.affiliation: Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA. Electronic address: aramer-tait2@unl.edu.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Ramer-Tait
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents; D001419:Bacteria / Q000145:classification / Q000235:genetics*; D002432:Cecum / Q000382:microbiology; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D004032:Diet; D005243:Feces / Q000382:microbiology; D005260:Female; D000069196:Gastrointestinal Microbiome / Q000235:genetics*; D041981:Gastrointestinal Tract / Q000382:microbiology; D005798:Genes, Bacterial; D005856:Germ-Free Life; D054884:Host-Pathogen Interactions; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D012407:rRNA Operon / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Altered Schaedler Flora; Antibiotics; Diet; Gut microbiota; Mice; qPCR