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Systematic optimization yields an efficient amplification and hybridization workflow that directly uses whole blood, bypassing nucleic acid extraction. The method tolerates ~24% whole blood, achieves 1.5 copies/μL sensitivity, shows no cross-reactivity, and matches sequencing with 100% concordance. Integrated with microfluidics, a closed-tube sample-in/result-out platform reduces total time to 122 min. 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\n[http://pubs.acs.org/journal/acsodf](http://pubs.acs.org/journal/acsodf)  Article   \nInnovative Rapid Detection System for Nondeletion-Type α‑Thalassemia via Whole-Blood Amplification and Microfluidic Technology  \nYan Wei, Yilian Zhao, Chao Ye, Mengru Xie, Xiaoxing Zhou, Li Youqiong, Gao Huiyuan, Jilin Qing, * Tan Tan, * and Zhizhong Chen *  \n Cite This: ACS Omega 2025, 10, 59345−59358  \nRead Online  \n\n|  |  |  |  |  |  |\n| --- | --- | --- | --- | --- | --- |\n| ACCESS   | Metrics & More |  |  Article Recommendations |  | *sı Supporting Information |\n\nABSTRACT: Thalassemia represents a major global public health challenge. This study developed an integrated detection system combining direct PCR(Dir-PCR) with reverse dot blot hybridization (RDB), enabling accurate and rapid diagnosis of nondeletion α -thalassemia (αWSα, αCSα, αQSα). The core innovation lies in the systematic optimization of the Dir-PCR-RDB methodology, establishing an efficient amplification and hybridization system capable of directly utilizing whole blood samples, thereby effectively bypassing conventional nucleic acid extraction steps. The optimized method tolerates approximately 24%(12 μL/50 μL) whole blood, achieves a sensitivity of 1.5 copies/μL, and demonstrates no cross-reactivity with other thalassemia variants. Clinical validation showed 100% concordance with sequencing results. Furthermore, by integrating this optimized system with microfluidic technology, we constructed a  \nclosed-tube “sample-in, result-out” platform that reduces total detection time to 122 min. This work not only provides an efficient and cost-effective solution for nondeletion α-thalassemia screening but also establishes a technological paradigm for rapid molecular diagnosis of other genetic variants and pathogens.  \n1. INTRODUCTION  \nThalassemia is one of the most common single-gene disorders worldwide,1 which poses a significant challenge to global health. Defects in the α and/or β-globin genes that constitute hemoglobin leads to instability and impaired oxygen-carrying capacity of hemoglobin, resulting in anemia. Approximately 4.4 out of every 10,000 live births are affected globally.2,3 Among its variants, nondeletional α-thalassemia (such as αWSα, αCSα, and αQSα) is highly prevalent in Southeast Asia. Therefore, there is a critical need for rapid and accurate diagnostic tools to enable timely intervention.  \nNucleic acid amplification technology serves as the foundation for thalassemia genetic diagnosis.4 However, conventional PCR methods are susceptible to inhibitors in whole blood samples,5 necessitating cumbersome nucleic acid extraction that not only increases detection time and cost but also limits their application in primary healthcare settings.6,7 The emergence of direct PCR(Dir-PCR) technology has effectively addressed this bottleneck by enabling direct amplification from whole blood.8,9 Reverse dot blot hybridization (Dir-PCR) provides a practical platform for parallel detection of multiple mutations.10  \nDespite the conceptual advancement, current Dir-PCR-RDB techniques for nondeletional α-thalassemia remain at an early  \nstage, with existing studies showing significant limitations. A mature detection system compliant with Clinical and Laboratory Standards Institute guidelines has yet to be established. The entire process involves multiple open-tube steps, making full-process automation and closed-tube operation difficult to achieve, while introducing contamination risks and demanding high operator proficiency. Additionally, current methods struggle to meet the high-throughput, highsensitivity and tolerance for high blood volumes requirements of clinical testing. Moreover, reliance on visual interpretation of results introduces subjectivity, hindering the development of widely applicable clinical solutions.  \nMicrofluidic technology, with its advantages of high integration, 11, 12 automation, 13 standardization, and","cbCaiuJQ8WE6sfTd","https://ap.wps.com/l/cbCaiuJQ8WE6sfTd","pdf",3705781,14,"English","# Abstract\n# Introduction\n# Methods\n## Sample Collection and DNA Extraction","[{\"question\":\"What diagnostic targets does the system detect?\",\"answer\":\"It detects nondeletion α-thalassemia genotypes: αWSα, αCSα, and αQSα.\"},{\"question\":\"How does the method avoid conventional nucleic acid extraction?\",\"answer\":\"The optimized Dir-PCR-RDB workflow directly amplifies from whole blood, bypassing nucleic acid extraction steps.\"},{\"question\":\"What performance and validation results are reported?\",\"answer\":\"The method tolerates about 24% whole blood, shows sensitivity of 1.5 copies/μL with no cross-reactivity, and clinical validation shows 100% concordance with sequencing results.\"}]","Innovative Rapid Detection System for Nondeletion-Type α-Thalassemia via Whole-Blood Amplification and Microfluidic Technology | PDF",1790697578,35]