{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T05:07:33Z","timestamp":1789016853550,"version":"build-2803163510"},"reference-count":50,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004944","name":"Naresuan University","doi-asserted-by":"publisher","award":["R2567B080."],"award-info":[{"award-number":["R2567B080."]}],"id":[{"id":"10.13039\/501100004944","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R2567B080."],"award-info":[{"award-number":["R2567B080."]}],"id":[{"id":"https:\/\/ror.org\/03e2qe334","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>The \u03b10-thalassemia Southeast Asian (--SEA) and Thai (--THAI) deletions comprise a significant global health burden, with high prevalence in Southeast Asia. Conventional methods like polymerase chain reaction (PCR) are limited in resource-poor settings due to complexity, time, and cost. This assay was developed as a rapid and simple option for point-of-care (POC) applications. Three assays specific for wild-type DNA, --SEA and --THAI deletions utilized recombinase polymerase amplification (RPA) for rapid DNA amplification at a constant low temperature of 37 \u00b0C, with a run time of 10 min, eliminating the need for a thermal cycler. The amplified products were detected using the highly specific CRISPR-Cas12a system and read visually via fluorescence or combined with a lateral flow assay (LFA) in 30 min. Successful engineering of a synthetic PAM site enabled Cas12a recognition of the --SEA target despite the absence of a suitable natural PAM. The platform demonstrated high accuracy in 74 blinded clinical samples, achieving a diagnostic accuracy of 98.20%, a sensitivity of 94.59%, and a specificity of 100% across 222 per-target evaluations. Detection from non-invasive samples achieved 80% to 100% accuracy, depending on the extraction method and readout format used. Our platform showed potential to meet several ASSURED criteria for diagnostic tests in resource-limited settings, demonstrating high potential for widespread use in genetic screening for \u03b10-thalassemia (--SEA) and (--THAI).<\/jats:p>","DOI":"10.3390\/biology15181579","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T16:46:04Z","timestamp":1788885964000},"page":"1579","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of Gap-Recombinase Polymerase Amplification Combined with CRISPR-Cas12a (Gap-RPA\/CRISPR-Cas12a) for Rapid Screening of \u03b10-Thalassemia Southeast Asian (--SEA) Deletion and Thai (--THAI) Deletion"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2715-0570","authenticated-orcid":false,"given":"Phanupong","family":"Changtor","sequence":"first","affiliation":[{"name":"Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7676-6407","authenticated-orcid":false,"given":"Siriphat","family":"Muangpa","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0989-865X","authenticated-orcid":false,"given":"Nonglak","family":"Yimtragool","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"81","DOI":"10.3390\/thalassrep14040010","article-title":"Thalassemia: Pathophysiology, Diagnosis, and Advances in Treatment","volume":"14","author":"Sadiq","year":"2024","journal-title":"Thalass. 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