TY - JOUR
T1 - A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice
AU - Gomes-Neto, João Carlos
AU - Mantz, Sara
AU - Held, Kyler
AU - Sinha, Rohita
AU - Segura Munoz, Rafael R.
AU - Schmaltz, Robert
AU - Benson, Andrew K.
AU - Walter, Jens
AU - Ramer-Tait, Amanda E.
N1 - Funding Information:
All authors are grateful for the technical expertise and skillful animal husbandry provided by Brandon White and all the staff at the UNL Gnotobiotic Mouse Facility. We also thank Dr. Michael Wannemuehler (Iowa State University) for providing the ASF strains and founder ASF-bearing C3H/HeN mice. This work was supported by a Career Development Award from the Crohn's and Colitis Foundation of America (grant # 3578), the National Institute of General Medical Sciences of the National Institutes of Health (1P20GM104320), and start-up funding from the University of Nebraska-Lincoln to ART. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
PY - 2017/4/1
Y1 - 2017/4/1
N2 - 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.
AB - 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.
KW - Altered Schaedler Flora
KW - Antibiotics
KW - Diet
KW - Gut microbiota
KW - Mice
KW - qPCR
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U2 - 10.1016/j.mimet.2017.02.003
DO - 10.1016/j.mimet.2017.02.003
M3 - Article
C2 - 28189782
AN - SCOPUS:85012300477
VL - 135
SP - 52
EP - 62
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
SN - 0167-7012
ER -