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The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Primer and probe sequences were strategically positioned at conserved regions in <jats:italic>psi<\/jats:italic> and <jats:italic>env<\/jats:italic> and adapted to subtype B&amp;C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict \u00a0the\u00a0specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The in silico analysis showed that the HIV-1 B&amp;C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&amp;C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&amp;C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (&gt;\u200995% certainty) at 60\u00a0\u00b0C. Quantification of 2\u20136 copies can be performed with\u2009&gt;\u200980% certainty. Lowering the annealing temperature to 55\u00a0\u00b0C slightly lowered the\u00a0specificity but prevented exclusion of samples with single mutations in the primer\/probe region.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12985-024-02300-6","type":"journal-article","created":{"date-parts":[[2024,1,31]],"date-time":"2024-01-31T16:02:30Z","timestamp":1706716950000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C"],"prefix":"10.1186","volume":"21","author":[{"given":"N. 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The studies from which the HIV-1 subtype B sequences originate have been reviewed by institutional review boards at UCSF, the Western Sydney Local Health District, which includes the West-Mead institute for Medical Research, the Research Ethics Committees at the Alfred and Avenue Hospitals in Melbourne, and the University of Melbourne Ethics Committee [\u2013]. The subtype C sequence studies were approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal and the institutional Review Board of Massachusetts General Hospital [, ].","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no conflict of interest.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"36"}}