Abstract Background Presence of contaminant DNA in molecular reagents, even in trace amounts, is potentially amplifiable and can lead to false positives and decreased sensitivity in molecular diagnostic tests. Contaminant DNA can come from a multitude of sources such as sample handling, plasticware, and from assay reagent themselves. The goal of this study was to develop an assay capable of detecting trace amounts of DNA in various reagents, thus allowing for removal of contaminant DNA and development of functionally DNA-free reagents. Methods A qPCR-based assay was developed for detection of trace quantities of DNA in biological samples. Universal qPCR primers towards the eukaryotic 18S ribosomal gene, the bacterial ribosomal 16S gene, and the bacterial plasmid Ori gene were used to cover a wide breadth of biological diversity. 40 reagent control and test replicates were subjected to SYBR Green qPCR for each primer pair. DNA contamination was determined by extrapolation from concomitantly run standard curves. This assay was used to measure contaminant DNA in various reagents, allowing for monitoring of trace quantities of DNA and for the development of reagents containing minimal amplifiable DNA. Results Positive control templates consisting of E. coli genomic DNA, human genomic DNA, and pUK19 plasmid serially diluted 1:10 showed linear amplification response, allowing for use as a standard curve. Implementation of various environmental controls including a dedicated PCR hood, strict cleaning procedures, sterile gowning, and UV treatment of handling materials allowed for a consistent threshold of non-amplification events in negative control sterile water. Preparations of DNA-free DNA polymerases, reverse transcriptase, and their respective reaction buffers, cofactors, and substrates as well as silica magnetic beads used for nucleic acid purification were demonstrated to contain less than 1 copy of prokaryotic DNA, less than 0.1 copies of eukaryotic DNA, and less than 10 copies of plasmid DNA per 40 PCR replicates. Conclusion These approaches demonstrate that trace amounts of DNA can effectively be monitored via a qPCR-based DNA contamination assay. This assay allows for the development and testing of functionally DNA-free reagents including enzymes, buffers, and magnetic beads, ultimately decreasing risks of false positives due to non-specific DNA contamination.
Caffarelli et al. (Wed,) studied this question.