A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice

Authors

João Carlos Gomez-Neto, University of Nebraska-Lincoln
Sara Mantz, University of Nebraska-Lincoln
Kyler Held, University of Nebraska-Lincoln
Rohita Sinha, University of Nebraska-LincolnFollow
Rafael R. Segura Munoz, University of Nebraska-LincolnFollow
Robert Schmaltz, University of Nebraska-LincolnFollow
Andrew K. Benson, University of Nebraska-LincolnFollow
Jens Walter, University of AlbertaFollow
Amanda E. Ramer-Tait, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2017

Citation

João Carlos Gomes-Neto, Sara Mantz, Kyler Held, Rohita Sinha, Rafael R. Segura Munoz, Robert Schmaltz, Andrew K. Benson, Jens Walter, Amanda E. Ramer-Tait , A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mimet(2017), doi: 10.1016/j.mimet.2017.02.003

Comments

Copyright 2017 Elsevier. Used by permission.

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.