PCR Inhibition Assay for DNA-targeted Antibiotics.

Abstract

DNA amplification by polymerase chain reaction (PCR) should be inhibited if the target for amplification region in the template DNA is nicked or cut. Based on this premise, we established a sensitive and differential assay using PCR to detect antibiotics that act on DNA. After template λDNA (10 pg) was incubated with antibiotics (10-20ng) at 37°C for 30 minutes in a 5μl reaction volume, a PCR assay (10μl reaction volume; 25-30 cycles) was performed under the conditions we modified, resulting in amplification of a 500 bp fragment of λDNA which was monitored by agarose gel electrophoresis. Among the several antibiotics examined, the anthracyclines, bleomycin, D-cycloserine and mitomycin C clearly inhibited the PCR amplification reaction, whereas actinomycin D and ofloxacin did not. Preincubation of template DNA in the presence of Fe+ + was necessary for bleomycin and cycloserine to exhibit marked inhibition of PCR. Mitomycin C exhibited the inhibition in the presence of DTT and Cu+. By contrast, non-DNA-acting antibiotics (200 ng) such as aminoglycosides, β-lactams, and macrolides showed no inhibition. The PCR-amplified fragment from λDNA was not degraded by incubation with the antibiotics (20 ng) that inhibited PCR. Furthermore, ethylacetate extracts of the cultured broths of actinomycetes proved to be suitable as samples for this PCR inhibition assay.