Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces

Abstract

Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS): A Pilot Study

“…[34]. 20 ng DNA template were used in a 10 μl reaction on a LightCycler 480 Instrument (Roche) according to the manufacturer’s instructions.…”

Critical Role for a Subset of Intestinal Macrophages in Shaping Gut Microbiota in Adult Zebrafish

“…We employed previously established primers that target species of the family Desulfovibrionaceae (which encompasses Lawsonia ; Bergström et al, 2012; Fite et al, 2004) and the genus Cetobacterium (Brugman et al, 2014), in addition to universal 16SrRNAgeneprimers for total bacteria (Mottawea et al, 2016; Yang et al, 2015). In line with the results obtained by deep sequencing, we observed a clear overabundance of the normally rare Desulfovibrionaceae in irf8 mutant guts, which was below detection in siblings (Figure 3C).…”

Absence of receptor guanylyl cyclase C enhances ileal damage and reduces cytokine and antimicrobial peptide production during oralSalmonellaTyphimurium infection

“…Bacterial DNA was isolated from fecal pellets using the QIAamp ® DNA Stool Mini Kit according to manufacturer’s instructions. The abundance of total bacteria and specific intestinal bacterial phylum, class and species was quantified by real-time PCR analysis by SYBR ® Premix Ex Taq ™ (Tli RNaseH Plus) on a StepOnePlus real-time PCR machine (Applied Biosystems, USA) using 16S primers published earlier (57, 58). The efficiencies of each primer were estimated (data not shown) and were found to be > 90%.…”