TITLE:
Number of PCR Cycles and Magnesium Chloride Concentration Affect Detection of tet Genes Encoding Ribosomal Protection Proteins in Swine Manure
AUTHORS:
Gunilla Schmidt, Jill Stiverson, Øystein Angen, Zhongtang Yu
KEYWORDS:
Porcine, Antibiotic Resistance, Assay, Optimization, Reaction-Mix
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.12,
September
16,
2014
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 MgCl2 concentrations were evaluated for their effect on the diversity and prevalence
of the tet genes that encode
ribosomal protection proteins (RPPs) in composted swine fecal samples using the
degenerate Ribo2_new_FW/Ribo2-RV primer pair. Four MgCl2 concentrations
and 3 cycle numbers were tested in a 4 × 3 factorial design. A clone library was
constructed for each PCR condition combination, 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 MgCl2 concentration combinations. The results suggest that 35 PCR cycles and 7 mM
MgCl2 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.