Development of a semiquantitative degenerate real-time pcr-based assay for estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase genes in complex bacterial samples

Abstract

We describe a new degenerate real-time PCR approach to simultaneously quantify phylogenetically different butyrate-producing bacteria based on the detection of butyryl-coenzyme A (CoA) CoA transferase genes. This pathway is present in numerically important groups of butyrate producers within the human colon, and thus this assay estimates the butyrate-producing ability of the microbiota.

FIG. 1.

Nucleic acid and amino acid sequence alignments of primer binding sites of degenerate primers BCoATscrF and BCoATscrR. Butyryl-CoA CoA transferases: R. hom., R. hominis A2-183; E. hal., E. hallii L2-7; A. cac. I, A. caccae L1-92; F. prau., F. prausnitzii A2-165. Putative acetyl-CoA hydrolase: D. haf., D. hafniense. 4-Hydroxybutyrate CoA transferases: C. klu., C. kluyveri; C. tet., C. tetani; A. cac. II, A. caccae L1-92; C. am., C. aminobutyricum (for accession numbers, see the text). Primer binding sites correspond to nucleotide positions 217 to 249 for primer BCoATscrF and 748 to 773 for primer BCoATscrR of the butyryl-CoA CoA transferase gene from R. hominis A2-183. (*) The sequence for primer BCoATscrR is given in reverse to enable comparison to the alignment. Amino acids encoded by the primer sequence are indicated. Arrows indicate primer direction. Black shading indicates positions identical to the butyryl-CoA CoA transferase consensus, and gray shading indicates matches due to degeneracy of the primers. I, inosine; N, A, C, G or T; S, C or G; W, A or T; Y, T or C.

FIG. 2.

Quantification of butyryl-CoA CoA transferase genes within samples from fermentors inoculated with human fecal slurries (start) from two donors grown at two pH values and in the presence of 0.6% peptide as described by Walker et al. (16). Data for quantification of bacterial groups based on FISH are taken from reference . Gray bars, F. prausnitzii group; black bars, Roseburia spp. and E. rectale group; hatched bars, other clostridial cluster XIVa bacteria; stippled bars, results of quantification by real-time PCR, expressed as the number of CoA transferase genes detected per 16S rRNA gene.