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Published on: July 13, 2019
Translating genomic exploration of the family Polyomaviridae into confident human polyomavirus detection
Sergio Kamminga1,2, Igor A Sidorov1, Michaël Tadesse1
1Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands.
Abstract:
The Polyomaviridae is a family of ubiquitous dsDNA viruses that establish persistent infection early in life. Screening for human polyomaviruses (HPyVs), which comprise 14 diverse species, relies upon species-specific qPCRs whose validity may be challenged by accelerating genomic exploration of the virosphere. Using this reasoning, we tested 64 published HPyV qPCR assays in silico against the 1781 PyV genome sequences that were divided in targets and nontargets, based on anticipated species specificity of each qPCR. We identified several cases of problematic qPCR performance that were confirmed in vitro and corrected through using degenerate oligos. Furthermore, our study ranked 8 out of 52 tested BKPyV qPCRs as remaining of consistently high quality in the wake of recent PyV discoveries and showed how sensitivity of most other qPCRs could be rescued by annealing temperature adjustment. This study establishes an efficient framework for ensuring confidence in available HPyV qPCRs in the genomic era.
Insights
This study validated and improved human polyomavirus (HPyV) quantitative PCR assays. We ensured reliable HPyV detection in the genomic era by optimizing assays for accuracy and sensitivity.
Area of Science:
- Virology
- Molecular Biology
- Bioinformatics
Background:
- Polyomaviridae are ubiquitous dsDNA viruses causing persistent infections.
- Human polyomaviruses (HPyVs) detection relies on species-specific qPCR assays.
- Genomic exploration challenges the specificity of existing HPyV qPCR assays.
Purpose of the Study:
- To evaluate the performance of published HPyV qPCR assays against extensive viral genomic data.
- To identify and correct problematic qPCR assays for accurate HPyV detection.
- To establish a framework for validating HPyV qPCR assays in the genomic era.
Main Methods:
- In silico analysis of 64 HPyV qPCR assays against 1781 PyV genome sequences.
- In vitro validation of identified problematic qPCR assays.
- Optimization of qPCR assays using degenerate oligonucleotides and annealing temperature adjustments.
Main Results:
- Several HPyV qPCR assays showed problematic performance in silico, confirmed in vitro.
- Assay performance was improved using degenerate oligonucleotides.
- 8 of 52 BKPyV qPCRs maintained high quality; sensitivity of others was rescued by annealing temperature adjustment.
Conclusions:
- A robust framework was established for validating HPyV qPCR assays.
- The study ensures confidence in HPyV detection methods amidst viral genomic discoveries.
- Optimized qPCR assays are crucial for accurate HPyV surveillance and research.
