1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays

Abstract Background Success of real-time TaqMan PCR (qPCR) in detecting pathogen targets and quantifying pathogen load is dependent upon frequent assay monitoring. This is due to i) the high degree of complementarity needed between primers / probes and genomic targets for assay accuracy and ii) natural pathogen variation and evolution. Failure to monitor and refine may result in false negativity or under quantification. Here we present a bioinformatics tool to identify potential problems resulting from newly discovered genomic mutations in primer/probe regions. Methods The tool performs an unbiased and automated search of the NCBI database, collects relevant genomic sequences based on user-defined Taxon-ID and executes a Python program to discard synthetic sequences. A profile of primer-probe sequence complementarity to targets is then generated. While the tool can be used for any microbe, here we present results for our laboratory’s cytomegalovirus (CMV) qPCR primer-probe analysis. In addition, our laboratory’s traditional approach utilizing alignment software was performed (download of all CMV sequences (~10,000) followed by iterative alignment building of these against our primers and probes). The amount of time to perform the automated and manual methods was recorded. Results The tool retrieved 8,732 sequences from NCBI and compared these to the CMV qPCR primers and probes. The tool found 2,501 alignments between the primers / probes and the downloaded genomic data (~15 minutes to finish (6 CPUs)). A total of 64% (1,624/2,501) of BLASTn alignments were exact matches between all primers / probes and viral genomic sequences. 17.5% (439/2,501) of alignments had 1 mismatch at either 5’ or 3’ terminus, and 1% (25/2,501) of alignments had two mismatches with the primers / probes. Similar results were found using a primarily manual approach (which took approx. 5 hours computing time and 20 hours of labor). Conclusion This new bioinformatics approach performed indistinguishably vs. a manual approach and did so in minutes rather than days. Both methods led to the conclusion that, by virtue of our design involving overlapping primers and probes, none of the identified mismatches are predicted to lead to false negativity or under quantification in our current CMV qPCR assay. Disclosures All authors: No reported disclosures.