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Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3\u2019 termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency \u22655% (2\u2009h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development.<\/jats:p>","DOI":"10.1038\/s42003-021-01751-9","type":"journal-article","created":{"date-parts":[[2021,2,20]],"date-time":"2021-02-20T04:32:45Z","timestamp":1613795565000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens"],"prefix":"10.1038","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5608-634X","authenticated-orcid":false,"given":"Iain J.","family":"MacLeod","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher F.","family":"Rowley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Essex","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,2,18]]},"reference":[{"key":"1751_CR1","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1093\/ilar\/ilx026","volume":"58","author":"R Carrasco-Hernandez","year":"2017","unstructured":"Carrasco-Hernandez, R., J\u00e1come, R., L\u00f3pez Vidal, Y. & Ponce de Le\u00f3n, S. 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Chan School of Public Health at the time this research was performed and is currently a co-founder, shareholder, and employee of Aldatu Biosciences, Inc, a diagnostics company that commercializes the PANDAA technology. Aldatu Biosciences, Inc had no role in the conceptualization, study design, data collection and analysis, and decision to publish or preparation of the manuscript. Patents relevant to this work include the following: US 10100349 B2 and EP 3052656 B1 (\u201cMethods of determining polymorphisms\u201d) on which I.J.M., C.F.R., and M.E. are the inventors and are assigned to the President and Fellows of Harvard College, Cambridge, MA.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"227"}}