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1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays
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 path...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6809443/ http://dx.doi.org/10.1093/ofid/ofz360.1649 |
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author | Sinha, Rohita Wissel, Mark Bartlett, Katelyn Grantham, James Kleiboeker, Steve |
author_facet | Sinha, Rohita Wissel, Mark Bartlett, Katelyn Grantham, James Kleiboeker, Steve |
author_sort | Sinha, Rohita |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6809443 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-68094432019-10-28 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays Sinha, Rohita Wissel, Mark Bartlett, Katelyn Grantham, James Kleiboeker, Steve Open Forum Infect Dis Abstracts 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. Oxford University Press 2019-10-23 /pmc/articles/PMC6809443/ http://dx.doi.org/10.1093/ofid/ofz360.1649 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Abstracts Sinha, Rohita Wissel, Mark Bartlett, Katelyn Grantham, James Kleiboeker, Steve 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title | 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title_full | 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title_fullStr | 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title_full_unstemmed | 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title_short | 1786. An Automated Method to Assess Oligonucleotide Primer and Probe Complementarity to Genomic Targets in Infectious Disease qPCR Assays |
title_sort | 1786. an automated method to assess oligonucleotide primer and probe complementarity to genomic targets in infectious disease qpcr assays |
topic | Abstracts |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6809443/ http://dx.doi.org/10.1093/ofid/ofz360.1649 |
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