Number of PCR Cycles and Magnesium Chloride Concentration Affect Detection of tet Genes Encoding Ribosomal Protection Proteins in Swine Manure

Abstract

PCR is routinely used in detection of antibiotic resistance genes including different classes of tet and erm genes. It remains unknown how PCR conditions affect detection of resistance genes in terms of genetic diversity and prevalence. In this study, numbers of PCR cycles and MgCl 2 concen-trations were evaluated for their effect on the diversity and prevalence of the tet genes that en-code ribosomal protection proteins (RPPs) in composted swine fecal samples using the degener-ate Ribo2_new_FW/Ribo2-RV primer pair. Four MgCl 2 concentrations and 3 cycle numbers were tested in a 4 × 3 factorial design. A clone library was constructed for each PCR condition combina-tion, and randomly selected clones were sequenced to determine the genetic diversity and relative distribution of RPP tet genes. Significant differences in genetic diversity and prevalence of tet genes were found among the tested cycle numbers and MgCl 2 concentration combinations. The results suggest that 35 PCR cycles and 7 mM MgCl 2 allow for optimal detection of the tet genes in swine feces using the Ribo2_new_FW/Ribo2-RV primer pair and that this combination should be used for further assay optimization and validation. These results also suggest that PCR conditions should be taken into consideration when PCR conditions are chosen for ecological studies of tet genes and when the results are interpreted.