Single-nucleotide primer correction significantly improves qPCR/RT-qPCR performance for adenovirus detection: a case study of the modified Jothikumar kit
DOI:
https://doi.org/10.69139/baw87c74Keywords:
AdV3 , adenovirus detection, myocarditis, SYBR Green, primer design, primer–template mismatch, qPCR , qPCR sensitivity, viral diagnosticsAbstract
Background: Primer–template mismatches are an important yet frequently underestimated cause of reduced sensitivity in qPCR assays. Even a single mismatch, particularly near the 3′ terminus, can impair polymerase extension, delay amplification, and increase the limit of detection. The widely used Jothikumar adenovirus qPCR assay is known to perform suboptimally for human adenovirus type 3 (AdV3) due to mismatches within the binding region of the forward primer.
Material and methods: In this study, we assessed how correcting a single-nucleotide mismatch affects qPCR performance for AdV3. Using SYBR Green–based qPCR and ten-fold serial dilutions of quantified viral DNA, we compared amplification efficiency, Cq values, linearity, and analytical sensitivity between the original primer and a modified version designed to restore full complementarity.
Results: The original primer showed poor amplification efficiency, a shallow standard-curve slope, and a substantially elevated detection threshold. In contrast, the corrected primer achieved near-optimal efficiency, excellent linearity, improved reproducibility, and at least a ten-fold lower limit of detection. Across matched dilution points, the modified primer amplified approximately twenty cycles earlier than the unmodified version, demonstrating a profound impact on assay sensitivity.
Conclusion: These results show that even minimal optimization of primer sequence can markedly improve diagnostic performance. Routine in silico evaluation and periodic reassessment of primer–template compatibility should therefore be standard practice, especially for genetically variable viral targets such as adenoviruses.
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Copyright (c) 2026 Maciej Baron, Piotr Lewandowski MD, Jakub Kancerek (Author)
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